1.1.1. LiAc Yeast Transformation
1.2.1. E. coli over night Culture
1.2.2. Preparation of Electro Competent E. coli
1.2.3. Electroporation Transformation of E. coli
1.2.4. Preparation of Heat Shock Competent E. coli
1.2.5. Heat Shock Transformation of E. coli
1.2.6. Glycerol Stock of E. coli
1.2.7. Crude Plasmid DNA Preparation by Alkaline Lysis
1.2.8. Ethanol/NaAc Precipitation of Plasmid DNA
1.2.9. Plasmid DNA Preparation by Alkaline Lysis:
1.2.10. Isopropanol Precipitation of Plasmid DNA
1.2.12. Polymerase Chain Reaction – PCR
1.2.13. Single Restriction Digest
1.2.15. Agarose Gel Electrophoresis
1.3. Mammalian Cell Culture Methods
1.3.1. Cell culture of 293, HeLa, MEF, stable transfected 293
Cell Lines
1.3.2. Freezing of Mammalian Cell Lines
1.3.3. Thawing of Mammalian Cell Lines
1.3.5. Lipofectamine Transfection of Mammalian Cell lines
1.3.6. Generation of Stable Cell Lines
1.4.1. Cytosolic Cell Extract Preparation
1.4.2. Protein Quantification (Bradford)
1.4.5. Electro Transfer Blotting on PVDF Membrane
1.4.7. Silver Staining of PAGE Gels
1.4.8. Stripping of PVDF Membranes
1.4.9. Immunoprecipitation of Proteasomes
1.4.11. Glycerol Gradient Centrifugation
1.4.13. Proteasome Activity Assay
1.4.14. Proteasome Activity Overlay Assay
1.4.15. Trichloro Acetic Acid Precipitation
1.4.16. Kinase Assays with COP9 Complex as Substrate
1.4.17. Detection of 32P g-ATP Phosphorylated Proteins
1.5. GFP, YFP and CFP: the Living Color Family
1.6. Confocal laser scanning microscopy
1.6.1. Fluorescence Microscopy of Fixed Cells
1.6.2. Live Cell Fluorescence Microscopy
1.6.3. Fluorescence Recovery after Photobleaching (FRAP)
studies
Solutions:
10 mL of SD -Trp medium are inoculated with the
appropriate yeast strain and incubated at 30°C while shaking at 200 rpm o/n. On
the next day OD at 600 nm is measured and the yeast culture is diluted with YPD
to OD600 0.1. A total volume of 50 mL diluted yeast culture is used
for further incubation. Every hour OD600 is measured until OD600
0.4 is reached (3 - 5 hours). Then the cell number is calculated with a Thoma
chamber. 2x107 cells/mL are sufficient for 10 transformations. The
yeast is then harvested by centrifugation at 3000 rpm for 5 minutes, the
supernatant is carefully removed and collected for autoclaving. The pellet is
resuspended in 25 mL sterile AD and again centrifuged at 3000 rpm for 5 minutes.
After removing of the supernatant the pellet is resuspended in 1 mL LiAc 100
mM. Excess of LiAc is removed by spinning the tubes for 15 seconds at full
speed in a tabletop centrifuge and carefully removing the supernatant. The
yeast pellet is brought to a final volume of 500 µL with LiAc 100 mM. Aliquots
of 50 µL are prepared. One 50 µL aliquot of this yeast suspension is used for
one transformation. 50 µL aliquots are again briefly centrifuged to pellet the
cells, the supernatant is removed and on top of the yeast pellet, layers of the
following transformation solutions are pipetted in following order: 240 µL 50 %
PEG, 36 µL LiAc 1 M, 3.3 µL of bacterial RNA (31 µg/µL), 70.7 µL sterile AD, 1
µg plasmid DNA (1µg/µL). The tube is then thoroughly mixed by vortexing for 1
minute until the yeast pellet is completely dissolved and placed for 30 minutes
in a 30°C water bath. The tube is then transferred to a 42°C water bath for 25
minutes in order to perform the heatshock. The transformation mix is then
briefly centrifuged for 15 seconds at 4 000 x g (7 000 rpm) in a table top
centrifuge, the supernatant is discarded and the pellet is resuspended in 1 mL
sterile AD. 50 µL of this transformed yeast suspension are plated on SD – Leu,
- Trp, - Ade plates and incubated at 30°C for some days.
Solutions:
For preparing a saturated E. coli over night culture,
5 mL LB broth containing antibiotics are inoculated with a single colony of the
desired bacteria strain in a 50 mL tube. Incubation is carried out at 37°C with
constant shaking at 200 rpm over night (o/n). After 12 hours the culture is in
the state of stationary phase, no more increase of biomass is observed.
Solutions:
10 mL LB broth are inoculated with E. coli HB101 and
an o/n culture are prepared. On the next day the o/n culture is diluted in 1 L
LB broth and incubation is continued for around 3 hours. Optical density (OD)
at 600 nm is measured after 1 hour respectively until OD 0.4 - 0.6 is reached.
By that means the bacteria are in the state of exponential phase. The bacteria
broth is then cooled down on ice for 15 – 30 minutes in order to stop the
bacteria from dividing. Harvesting of bacteria is carried out by centrifugation
at 2500 x g, 4°C for 15 minutes (centrifuge:
Sorvall RC-5B), the supernatant is removed
and collected for autoclaving. The bacteria pellet is resuspended in 1 L
sterile AD (4°C) and again centrifuged at 2500 x g for 15 minutes at 4°C. The
supernatant is discarded and the pellet is resuspended in 500 mL sterile AD
(4°C) and again centrifuged at 2500 x g for 15 minutes at 4°C. The supernatant
is discarded and the pellet is resupended in 20 mL 10 % sterile glycerol (4°C)
and again centrifuged at 2500 x g for 15 minutes at 4°C. The supernatant is
discarded and the pellet is resuspended in 2 - 5 mL 10 % sterile glycerol
(4°C). 50 µL aliquots of this bacteria solution are prepared and quickly frozen
in liquid N2. Aliquots are stored at -80°C and can be used for 1
year.
Solutions:
50 µL electro-competent bacteria aliquot is thawed on
ice and transferred into prechilled electroporation cuvettes, 0.5 - 1 µg
plasmid DNA (or 5 - 10 µL of ligation mix) is added (0.1 cm). The surface of
the cuvette was dried prior to electroporation. Electroporation parameters
(power supply : voltage: 1.25 – 1.9 kV, capacity: 25 µF, resistance: 200
Ω. A successful electroporation is achieved with a time constant between 4
– 5 ms. After electroporation the bacteria are resuspended in 1 mL of SOC
medium and transferred in a 3059 Falcon tube. The bacteria are then incubated
at 37°C at 200 rpm for 1 hour. After recovery the transformed bacteria are briefly centrifuged, resuspended in 100 µL SOC
medium and the suspension is plated on LB agar plates containing appropriate
antibiotics. The plates are incubated over night at 37°C.
Solutions:
10 mL LB broth are inoculated with E. coli HB101 or
another appropriate strain such as DH5a and an o/n culture is prepared. On the next day the o/n culture is
diluted in 1 L LB broth and incubation is continued for around 3 hours. Optical
density (OD) at 600 nm is measured after 1 hour respectively until OD 0.4 – 0.6
is reached indicating that the bacteria are in the exponential phase of growth.
The bacteria broth is then cooled down on ice for 15 – 30 minutes in order to
stop the bacteria from dividing. Harvesting of bacteria is carried out by
centrifugation at 2500 x g, 4°C for 15 minutes (centrifuge: Sorvall RC-5B). The
supernatant is removed and collected for autoclaving. The pellet is resuspended
in a total volume of ~ 50 mL (36 mL TSS buffer + 12
mL of glycerol, 1/20 volume of diluted culture). Aliquots of 200 µL are
prepared, quickly frozen in liquid N2 and stored at -80°C.
Solutions:
An aliquot of 50 µL heat shock
competent E. coli (DH5a, stored at –80°C) is quickly
thawed in the palm and left for 10 minutes on ice. The aliquot is then
transferred into a pre-chilled Falcon 2059 tube. About 1 µg of DNA (1 - 2.5 µL)
is pipetted into the competent bacteria and the tube is gently swirled. After
20 minutes of incubation on ice the tube is placed in a prewarmed 42°C water
bath for 90 seconds without moving the tube. In order to cool down the sample,
the tube is immediately placed on ice for 1-2 minutes. Thereafter 800 µL of SOC
medium are added to the transformation mix and the bacteria are incubated for 1
hour at 37°C and 200 rpm to allow recovery from the heat shock and start
expression of the selection gene. Plating: For simple retransformations 100 µL
are plated on appropriate LB (Luria Bertani) agar plates (prewarmed to 37°C)
supplemented with the appropriate antibiotics (kanamycin 25 µg/mL or ampicillin
100 µg/mL). For clonings after a ligation the whole bacterial suspension is
used by pelleting the bacteria of the transformation mix by briefly and gently
spinning down, discarding the supernatant and resupension of the pellet in 100
µL SOC medium (or the reminder of the supernatant). This concentrated
suspension containing all the bacteria is then plated on LB agar plates
containing appropriate antibiotics. The plates are incubated over night at
37°C.
Solutions:
For a -80°C E. coli glycerol stock, 400 µL of an E. coli o/n culture are mixed thoroughly
with 100 µL 87 % glycerol in a 1.5 ml reaction tube and stored at -80°C. For
inoculation of fresh LB broth, a yellow 200 µL tip is plunged into the frozen
(!) stock and then pipetted up and down in the LB broth in order to thaw and
resuspend the bacteria.
Solutions: Qiagen Plasmid Preparation
Kit
50 mL Falcon tubes filled with 5 mL of LB broth
containing appropriate selective antibiotics (ampicillin: 100 µg/mL or
kanamycin: 25 µg/mL) are inoculated with one colony of the desired transformed
E. coli strain and incubated o/n at 37°C with vigorous shaking at 200 rpm. On
the next day, a glycerol stock is made and then the cells are harvested with
centrifugation at 6000 rpm? for 15 minutes at 4°C (centrifuge: Heraeus model
Biofuge Primo R), the supernatant is removed and collected for autoclaving.
Leftovers of LB broth can decrease the yield of plasmid DNA, therefore the
Falcon tubes are placed upside down on a paper towel for some minutes in order
to remove as much LB broth as possible. The E. coli pellet is resuspended in
250 µL of Resuspension buffer, transferred to 1.5 mL reaction tubes, then 250
µL of Lysis buffer are added and the lysis mix is carefully inverted 5 – 6
times until the solution is viscous. Shearing of genomic DNA by heavy shaking
should be avoided. After 5 minutes incubation time at room temperature 250 µL
of Neutralization buffer are added and again the mix is carefully inverted
several times until a white precipitate of genomic DNA and proteins is visible.
The white precipitate is pelleted by centrifuging at 6000 g for 15 minutes at
4°C. The supernatant containing plasmid DNA is collected without perturbing the
pellet and centrifuged a second time to quantitatively remove genomic DNA
precipitate. The collected supernatant is then subjected to isopropanol
precipitation. The unpurified plasmid DNA solution is mixed with 0.7 volumes of
isopropanol. After 5 minutes of incubation at room temperature, precipitated
plasmid DNA is recovered with centrifugation at full speed, 16 000 x g, at 4°C
for 30 minutes (centrifuge: Heraeus Sepatech Megafuge 1.0 R), the supernatant
is removed and discarded. The plasmid DNA pellet is washed with 1 mL 70 % EtOH
(4°C) without disturbing the pellet and centrifuged for another 5 minutes at
full speed at 4°C and the supernatant is quantitatively removed. The pellet is
dried for 5 min at RT and dissolved in 20 µl AD or 1x TE buffer. In some cases
this DNA-solution was further purified by an EtOH/NaAc precipitation. The crude
preparation of plasmid DNA can be subjected to restriction digest analysis and
sequencing but cannot be used for transfection experiments of mammalian cells.
In the latter case, the crude DNA solution was purified by Qiagen DNA
purification columns.
Solutions:
The DNA solution or in some cases the restriction
digest sample was mixed with 1/10 volume of NaAc, 2.5 volumes of 100 % ethanol
(-20°C) and was quickly transferred to -70°C for 5 minutes incubation. Then the
plasmid was collected by centrifugation for 30 minutes at full speed, 16 000 x
g, 4°C (centrifuge: Heraeus Sepatech
Megafuge 1.0 R), the supernatant was removed
by suction. The pellet was washed with 1 mL 70 % ethanol (4°C) and again
centrifuged for 5 minutes at 14 000 rpm, 4°C and the supernatant was
quantitatively removed by suction. The pellet was air-dried for some minutes
prior to elution in an appropriate volume of AD or 1x TE buffer (to reach a
final concentration of about 1 µg/µl).
Solutions:
For midi (maxi) DNA preparations the QIAGEN Plasmid
Midi (Maxi) kit was used (Cat.no.12143, 12163). A 250 mL (500 mL) shaking flask
filled with 50 mL (100 mL) LB broth containing appropriate selective
antibiotics (ampicillin 100 µg/mL, kanamycin 25 µg/mL) were inoculated with the
desired transformed single colony of E. coli strain and incubated o/n at 37°C
with vigorous shaking at 200 rpm. The cells were harvested by centrifugation at
6000 g for 15 minutes at 4°C (centrifuge: Heraeus model Biofuge Primo R), the
supernatant was removed and collected for autoclaving. Leftovers of LB broth
can decrease the yield of plasmid DNA, therefore the centrifugation buckets
were placed upside down on a paper towel for some minutes in order to remove as
much LB broth as possible – remnants of LB on the walls of the tubes were
removed by suction. The E. coli pellet was resuspended in 4 mL (10 mL) of
Resuspension buffer P1, 4 mL (10 mL) of Lysis buffer (P2) were added and the
lysis mix was carefully inverted 5 – 6 times. After 5 minutes incubation time
at room temperature 4 mL (10 mL) of Neutralization buffer were added, again the
mix was carefully inverted several times until a white precipitate of genomic
DNA and proteins were visible and the neutralized lysate was incubated on ice
for 20 minutes. The white precipitate was pelleted by centrifuging at 6000 g
for 15 minutes at 4°C. The supernatant containing plasmid DNA was collected
without disturbing the pellet and centrifuged a second time 6000 g for 15
minutes at 4°C. In the mean time a Quiagen tip 100 (500) was equilibrated by
applying 4 mL (10 mL) of Equilibration buffer on the matrix of the tip, the
buffer entered the resin only by gravity flow. The supernatant of the
centrifugation was applied on the columns and entered the column by gravity
flow. 2 Washing procedures were performed with 10 mL (30 mL) of
Solutions:
The eluted midi (maxi) plasmid DNA solution was mixed
with 0.7 volumes of isopropanol (room temperature) and immediately centrifuged
for 30 minutes at full speed, 17 000 x g, 4°C (centrifuge: Heraeus Sepatech Megafuge 1.0 R). The supernatant is carefully removed. The DNA pellet is washed with 2
mL (5 mL) 70 % ethanol (room temperature) without disturbing the pellet and
another centrifugation step is carried out for 10 minutes at full speed, 4°C,
the supernatant is quantitatively removed by suction. The pellet is air dried
and dissolved in 100 µL AD. DNA yield
is quantified and the plasmid DNA is diluted to a final working concentration
of 1 µg/µL.
DNA exhibits a specific absorption maximum at 260 nm,
which is used for DNA quantification by an UV/VIS spectrophotometer (Pharmacia
Biotech Ultrospec 2000). An OD at 260 nm of 1 refers to a DNA amount of 50
µg/mL. An appropriate dilution (1:200 after midi or maxi prep) of the DNA
sample is necessary to stay in the linear range (0.1-1) of this quantification
method. The purity of DNA concerning the presence of residual proteins can be
calculated by the ratio of OD260/OD280. The aromatic amino acid tryptophan
exhibits an absorption maximum at 280 nm, which refers to the relative amount
of protein in the sample. An OD260/OD280 ratio of 1.8 – 2.0 is desired – which
is typical for pure DNA samples resulting from the shoulder of DNA-absorbance
at 280 nm.
Solutions:
For a 100 µL PCR reaction, following solutions are
mixed in a thin walled reaction tube (BioRad): 85 µL AD, 10 µL Pfu buffer 10x,
1 µL dNTP mix 2 mM, 1 µL forward primer 10 pmol/µL, 1 µL reverse primer 10
pmol/µL, 500 ng (1 µL) template DNA, 1 µL Pfu polymerase (2.5 U). Handling and
pipetting of PCR steps are carried out with gloves in order to prevent
contaminations. The reaction tubes arere placed in the hot (94°) PCR block
(Perkin Elmer Gene Amp PCR System 2400) and the following program is used:
step 1 10
minutes 94°C (denaturation)
step 2 1 minute
94°C (denaturation)
step 3 1 minute
56°C or other appropriate annealing temperature – about 2-4 °C below the
calculated melting temperature (annealing)
step 4 1.5 minutes/ 1kb 72°C (elongation)
step 5 5 minutes
68°C
step 6 Ą 4°C (cooling)
30 cycles of steps 2-4 are carried out. After finished
PCR, 6x DNA sample buffer is added to the PCR samples to inactivate the polymerase.
Amplified PCR products are resolved by 1 % agarose gel electrophoresis.
Restriction digest of 20 µL volume: 17 µL of Aqua
dest. (nuclease free) are mixed with 2 µL appropriate 10x restriction enzyme
buffer, 1 µg (1µg/µL) of DNA added and mixed well. Then 1 µL (10 U) of
restriction enzyme (-20°C) are added and again mixed well by swirling and
spinning down. After incubation time of 1 hour at 37°C in a water bath (or
other appropriate temperature according to manufacturers protocol) DNA
fragments can be resolved by agarose gel electrophoresis.
Solutions:
For a ligation reaction of total 10 µL, the following
solutions are mixed: 100 – 200 ng linearized purified vector DNA, an equimolar
or slightly higher amount of purified insert DNA, 1 µL T4 ligase buffer 10x,
0.5 µL T4 ligase and AD to a total of 10 µl. As a ligation control, another ligation
reaction missing the insert DNA is prepared, instead of the insert DNA the
equivalent volume of AD is added to the solutions. The ligations are either
incubated at 16°C over night or at room temperature for 4 hours. 5 µL of each
ligation sample are used for heat shock transformation or electroporation.
Solutions:
Typically, 1 % agarose gels are poured by heating up 1
g agarose / 100 mL 1 x TAE until agarose is completely dissolved and no smears
are visible. The hot agarose solution is cooled down to about 55°C and 5 µL
ethidium bromide solution are added and gently mixed to avoid formation of
bubbles. Then, the agarose solution is poured immediately into the gel cast
cassette, appropriate combs are placed in the solution and abundant bubbles are
pushed away from the slots by pipette tips. After about 30 minutes,
polymerization is finished and the agarose gel can be used for electrophoresis.
The agarose gel is placed in the electrophoresis chamber (BioRad) with the
slots facing the cathode and 1x TAE is added until the gel is sufficiently
covered by running buffer. If necessary, the slots are rinsed with running
buffer by pipetting up and down some running buffer in order to remove
leftovers of agarose slurry. DNA samples are mixed with 6x DNA sample buffer
(5:1) and loaded in the slots. After loading, electrophoresis is started
immediately in order to prevent diffusion of sample into the surrounding gel.
Electrophoresis parameters (power supply BioRad Power PAC 2000): 100 – 120 V
for at least 30 minutes depending on the DNA fragment size supposed being
resolved. Resolved DNA fragments are visualized under UV transillumination
(transilluminator: 260 – 340 nm range) and documented with a BioRad gel doc
system. For clonings care is taken that the DNA is not exposed too long to UV
light since UV light can irreversibly damage DNA. DNA fragments are excised
with a clean razorblade and gel extraction is carried out as described below.
Solutions:
For gel extractions usually the QIAquick gel
extraction kit is used (Qiagen, Cat.no. 28704). An excised agarose gel slice is
weighed to calculate the approximate gel slice volume (1 g = 1 mL) and
transferred into an reaction tube. 3 volumes of solubilization buffer are added
and incubated in a 50°C waterbath until the gel piece is completely dissolved.
1 gel volume isopropanol is added and mixed. The solution is then loaded on the
column and centrifuged for 1 minute at full speed (centrifuge: Eppendorf
centrifuge 5415 D), the flow through is discarded. 750 µL of wash buffer are
pipetted to the column and centrifuged under the same conditions as above, the
flow through is discarded and the column is placed in a new microreaction tube.
For elution of extracted DNA, 20 µL of elution buffer are directly applied on
the matrix, incubated for 1 minute at room temperature and centrifugation as
above is carried out. As an alternative to the Qiagen gel extraction kit,
sometimes GenElute columns (Sigma, cat. no. # 5-6500) are used, which are
equilibrated by loading of 100 µl 1x TE buffer, followed by brief
centrifugation (1 min at full speed), addition of the gel slice to the column
and centrifugation at full speed for 10 min. The resulting flow through (about
40 µl) contains about 90 % of the DNA, while the agarose remains in the
column.
Solutions:
293 cells (immortalized human embryonic kidney cells,
adherent), HeLa (human cervix carcinoma cells, adherent) and MEF (mouse
embryonic fibroblasts, adherent) are cultured in DMEM complete medium, in
addition to this DMEM complete medium, the medium for stable 293 cell lines
with a genomic integration of a neomycin-resistance plasmid containing the gene
of interest, contains 500 µg/mL G418 as permanent selection agent. Cells are
cultivated in 75 cm2 tissue culture flasks at 37°C, in a humidified
(90 %) and CO2 (5 %) containing atmosphere. All cell types are
trypsinized and passaged every second or third day in proportion of 1:4 or 1:6,
respectively. For passaging the cell culture medium is removed, about 13 mL of PBS
def. (room temperature) are used to wash the cells. PBS def. is removed as well
and about 3 mL trypsin solution (37°C) are pipetted into the flask and spread
evenly all over the bottom. Excess of trypsin is removed and the flask is
placed in the incubator for around 3 minutes. With an inverse microscope (Nikon
TMS F 4x or 10x objective) it is checked if the cells are already detached and
round in shape. Trypsinized cells are gently resuspended by several times
up/down pipetting in 8 mL (to 12 ml – according to the desired passaging ratio)
complete DMEM and 2 mL are transferred into a new tissue culture flask
containing 13 mL of DMEM complete or DMEM complete including G418. Tissue
culture flasks prepared for MEFs have to be gelatinized prior to passaging as
follows: 15 mL of 1 % gelatin solution (autoclaved and sterile filtered, 37°C)
are pipetted in a 75 cm2 tissue culture flask and incubated at 37°C
for 30 minutes, the gelatin solution is discarded afterwards. After this
procedure, passaging is performed as usual.
Solutions:
293, HeLa, MEF, stable transfected
293 cell lines are passaged as described above, seeded in 75 cm2
cell culture flasks and grown until 100 % confluency is reached. The cells are
trypsinized as described above, resuspended in DMEM complete medium (4°) and
centrifuged at 1000 rpm (400 g), 4°C for 5 minutes in a cell centrifuge with a
swing-out rotor. The supernatant is removed and the cell pellet of one 75 cm2
cell culture flask is resuspended in 2 mL freeze medium (4°C). 1 mL of
resuspended cells are pipetted in pre-chilled cryotubes, placed in an
isopropanol-box (“Mr Freeze”, 4°C) and then transferred to -80°C in order to
freeze the cells in a controlled and slow procedure. After 24 hours the cell
aliquots are transferred and stored in the liquid nitrogen tank at -196°C.
A cell aliquot of 293, HeLa, MEF or stable transfected
293 cell lines stored in liquid nitrogen is quickly thawed in a 37°C water bath
with constant moving to prevent local overheated spots warmer than 4°C. The
thawed cell suspension is resuspended and diluted in about 20 mL DMEM complete
medium and the cells are centrifuged at 1000 rpm for 5 minutes at 4°C. The
supernatant is removed by suction, the cell pellet is resuspended in DMEM
complete medium or supplemented with G418 and the cells are seeded in 75 cm2
cell culture flasks.
Solutions:
Preparation: 293 or HeLa cells are passaged and seeded
at a density of 500.000 cells/6 well (40 % confluence). After one day (80 %
confluence) transfection is carried out. Transformation mix: 2 µg of DNA (1
µg/µL) are diluted in 60 µL of aqua dest. in a
polystyrene tube and 9 µL of 2 M CaCl2 are added and mixed,
71 µL of HeBS are added dropwise by unscrewing a P200 Gilson Pipette while
constantly vortexing at half speed. After incubation of 5 minutes at room
temperature the solution is pipetted dropwise as evenly as possible on the
surface of the cell culture dish. Once the DNA/calcium-precipitates get in
contact with the cells the tissue culture dish should not be moved a lot.
Normally the proteins of interest are expressed sufficiently after 24 hours of
transfection (when CMV promoter containing plasmids are used).
Solutions:
Preparation: 293 or HeLa cells are passaged and seeded
at a density of 500,000 cells/6 well (40 % confluence). After one day (80 %
confluence) transfection is carried out. For transformation of one 6-well 293
(HeLa) cells, 100 µL DMEM Ć medium is mixed with 1 µg
(1.5 µg) plasmid DNA and 4 µL (5 µL) lipofectamine Plus reagent and incubated
for 15 minutes at room temperature. 100 µL of DMEM Ć are mixed with 3 µL (3 µL) lipofectamine reagent
added to the incubated solution and mixed gently. The lipofectamine/DNA
solution is further incubated at room temperature for 15 min. Thereafter, the
transformation mix is diluted with 800 µL DMEM Ć. The culture medium of 293 or HeLa cells is removed
by suction, the cells are carefully washed once with at least 1.5 mL DMEM Ć medium, the wash DMEM is removed and the
transformation mix is carefully added to the cells. The 293 (or HeLa) cells are
incubated with the transformation mix in the incubator for 3 hours (7 hours).
After the transformation period the transformation medium is removed and
replaced by DMEM complete medium. 1 day after transfection, cells are
investigated.
293 cells are seeded at a density of 500,000
cells/6-well. The next day CaCl2 transfection with linearized and
EtOH/acetate precipitated plasmid DNA is carried out as described above. 1 day
after transfection cells are trypsinized and reseeded in a 10 cm cell culture
Petri dish (about 60 cm2) and DMEM complete medium is exchanged with
DMEM complete medium containing 900 µg/mL G418. About 2 weeks later, colonies
that survived the G418 selection are checked by fluorescence microscopy for the
expression of the transfected GFP-fusion protein and positive colonies are
picked under the microscope (4x magnification objective) under sterile laminar
flow by scraping them off with the tip of a pipette, careful suction of the
colony and placement into a well of a 24-well plate containing the selection
medium. 1 d after the isolation, the adherent colony is separated into single
cells by trypsinization and reseeding of the cell suspension in the same well.
After reaching confluence, the cells are further expanded up to 75 cm2
flasks and aliquots are frozen in liquid nitrogen.
Solutions:
Typically cells are lysed with 0.2 % NP 40, protease
inhibitors in PBS. Incubation of some minutes is enough to achieve effective
lysis. Destruction of the cytoplasm membrane is checked with the inverse
microscope (10 x magnification), intact nuclei are visible whereas the
cytoplasmic membranes disappear. After lysis, cell extracts are centrifuged for
15 minutes at full speed, 17 000 x g at 4°C (centrifuge: Heraeus Sepatech
Megafuge 1.0 R) to get rid of cytoplasm membrane parts and nuclei.
Solutions:
96-well format: Generation of BSA standard curve: 0 –
5 µg of BSA respectively are pipetted in a well respectively. Cell extract
samples are diluted e.g. 1:10 and an appropriate amount is used for
quantification in order to stay in the linear range of the assay. 150 µL of diluted
BioRad Coomassie Reagent (5x) are added to each well and incubated for some
minutes. Absorbance at 595 nm is then measured with an Elisa reader (SLT Lab
instruments 340 ATTC). The BSA standard curve is evaluated in MS Excel.
According to the equation after linear regression analysis (for the linear
range, usually between 0 and 4 µg protein), protein amounts are calculated.
Solutions:
Preparation: An appropriate amount of agarose beads
with covalently bound antibody (about 15 µl of a 50 % slurry are necessary for
one immunoprecipitation) are washed twice with 1 mL PBS (4°C) and centrifuged for
1 minute at full speed in a table top centrifuge (centrifuge: Heraeus Sepatech
Megafuge 1.0 R). Blocking: the agarose beads are suspended in 1 mL 1 % BSA in
PBS solution and incubated while rotating for 1 hour at 4°C, in order to block
unspecific protein binding sites and therefore to increase the specificity. The
beads are centrifuged as above and the supernatant is removed.
Immunoprecipitation: cell extract and PBS (4°C) are added to the blocked beads
and PBS (4°C) is added to a final volume of 800 µL and incubated while rotating
for 1 hour at 4°C. Washing procedure: 4x washing with PBS (4°C) and
centrifugation are performed as described above. After the third time washing
the resuspended beads are transferred to a new reaction tube in order to prevent
that proteins that bound unspecifically to the tube are eluted by SDS-buffer.
20 µL of 1x SDS sample buffer are pipetted to the beads and proteins are
detached from the beads by heating to 95°C for 5 minutes. After centrifugation
for 3 minutes at full speed in a table top centrifuge, the supernatant is used
for SDS PAGE (or stored at -80°C for later electrophoresis and Western
blotting).
Solutions:
For 2
SDS-PAGE gels (6 cm x 9 cm x 0.015 cm) 10 mL of separating gel are prepared. The
following table contains the volumes of solutions according to 7.5 %, 10 % and
12.5 % SDS-PAGE gels. H2O, solution A and solution B are mixed
(preventing air bubbles), then APS and TEMED are added in order to start
polymerization. The separating gel solution is mixed briefly and is filled in
the prepared gel cast system (BioRad Mini Gel system), about 5 cm high (to
about 0.5 – 1 cm below the position of the bottom of the sample comb). A layer
of butanol is pipetted on top to exclude oxygen from the surface since the
presence of oxygen disables polymerization. After about 30 minutes
polymerization is finished, the butanol layer is poured off and the stacking
gel is layered on top of the separation gel, immediately (to prevent drying of
the gel).
For 2 stacking gels 10 mL are prepared. According to
the next table H2O, solution Ast and solution B are mixed, and then
APS and TEMED are added in order to start polymerization. The solution is
pipetted on the separating gel to the upper edge of the glass plates (until
overload, bubbles are swept away like this) and then the combs are placed
between the glass plates avoiding air bubbles. Again, polymerization is
finished after 30 minutes, the SDS PAGE gel is then ready for use.
stack SDS
PAGE |
|
Solution |
4 % |
H2O [mL] |
6.1 |
sol. Ast [mL] |
2.5 |
sol. B [mL] |
1.3 |
APS 10 % [µL] |
50 |
TEMED [µL] |
15 |
Protein samples are mixed with 6x protein loading dye
to a final concentration of 1x protein loading dye. Samples are boiled for 5
minutes at 95°C, and then briefly centrifuged for 2 minutes at full speed in an
eppendorf table top centrifuge. After polymerization, the gel sandwich is taken
out of the gel cast and placed in the electrophoresis tank, adding 1x running
SDS running buffer in the inner and outer chamber, the combs are removed and
the slots are cleansed from half-polymerized gel pieces by pipetting up and
down some running buffer. Then protein samples and prestained protein marker
are loaded on the gel. Electrophoresis is started immediately to prevent
diffusion of the samples into the surrounding gel area. Electrophoresis
parameters: 30 mA per gel for about 1 hour, depending on how big the resolved
proteins were.
Solutions
Activation of PVDF membrane (Millipore, Immobilon Cat.
No. #IPVH20200): in order to make the PVDF membrane hydrophilic it has to be
soaked in methanol 100 % for 15 seconds, then rinsed in aqua dest. for 2
minutes and equilibrated in Western transfer buffer for at least 3 minutes. The
blotting sandwich is built as follows: one blotting sponge lying on blotting
cassette soaked in western transfer buffer, two 3MM whatman filterpapers soaked
in western transfer buffer, the activated PVDF membrane, the polyacrylamide
gel, two 3MM whatman filterpapers soaked in western transfer buffer, one
blotting sponge soaked in western transfer buffer. After each layer possible
bubbles are removed by rolling a plastic pipet on top of the current layer. The
blotting cassette (BioRad) is then closed and placed in the transfer chamber
(orientation overview: cathode – / gel / membrane / anode +). Blotting parameters:
150 mA per blotting cassette/gel for 2 hours.
Solutions:
·
Ponceau S
solution: 0.1 % (w/v) Ponceau S in 5 % (v/v) acetic acid
·
Blocking
solution: 5 % skimmed milk powder in PBS def.,0.1 % Tween20
·
·
Antibody
solution: primary or secondary antibody diluted in 1 % skimmed milk powder in
PBS def., 0.1 % Tween20
·
Primary
antibodies: aIKK2 (1:500, mouse IMGENEX), aJAB1/CSN5 (1:10 000, rabbit,
·
Secondary
antibodies coupled with horseradish peroxidase (HRP) amouse (1:5000, Amersham-Pharmacia), a rabbit (1:5000, Amersham-Pharmacia)
·
NaN3
solution 20 % (w/v) 200x in AD
·
ECL:
(Amersham), ECL plus (Amersham)
·
Pierce
Super signal West pico (Cat. No. #34080)
·
Pierce
Super signal West femto (Cat. No. #34095)
The PVDF membrane is taken out of the
transfer sandwich, possible gel leftovers sticking on the membrane are removed
and the membrane is incubated in Ponceau S solution while gently shaking for 5
- 10 minutes. After this, the Ponceau S solution is removed and collected for
reuse, the membrane is destained by rinsing with AD until the bands can be
clearly seen. At this point, desired lanes can be separated by cutting and
trimming the membrane with a sharp scalpel. The stained membrane is scanned on
a regular computer-scanner to assess equal protein loading (respectively for
normalization purposes). Then the membrane is incubated in blocking solution
for 30 minutes while gentle shaking to block unspecific binding sites.
Appropriate primary antibodies are diluted in 5 – 10 mL antibody solution in a
50 mL Falcon tube. In order to cover the membrane evenly with antibody
solution, the membrane is placed on the inner wall of the 50 mL Falcon tube
after the blocking step with the protein side facing the inner side and the
antibody solution and incubated on a rotor for 1 hour at 4°C. For reuse the
antibody solution is supplemented with NaN3 20 % (1:200) to a final
concentration of 0.1 % NaN3 to prevent bacterial growth and stored
at 4°C. The membrane is rinsed twice shortly with about 20 mL wash solution,
then washed twice with about 100 mL wash solution for 5 minutes and washed once
with about 100 mL for 15 minutes. After the last washing step, the antibody
solution containing the secondary antibody is incubated under the same
conditions as above for 1 hour. The washing protocol is the same (2 x rinsing,
2 x 5 minutes, 1 x 15 minutes). Detection of immunoblotted proteins is done by
chemiluminescence reaction with substrate solutions provided by Pierce or
Amersham-Pharmacia. The membrane is incubated with the substrate solutions
according to the manufacturer’s protocol. (ECL: (amersham) mixing ratio of solution
1 and 2 1:1 incubation time 1 minute, ECL plus mixing ratio of solution A and B
1:40, incubation 5 minutes, Pierce Super signal West pico: mixing ratio of
solution 1 and 2 1:1 incubation time 5 minutes, Pierce Super signal West femto
mixing ratio of solution 1 and 2 1:1 incubation time 5 minutes). The membrane
is taken out of the solution and is placed in a plastic wrap avoiding wrinkles
of the wrap. Then excess of substrate solution was removed by streaking from
the outside with a paper towel. Depending on signal intensity the blot is
exposed to Kodak X-OMAT or BioMax films or a CCD-camera based detection system
(LumiImager, Roche) is used for detection of chemiluminescence. The distances
of the protein markers from the top of the separation gel are measured and used
for generating a standard curve of the molecular weights (using the correlation
between the log of the MW and the migration distance; calculations and
regression analysis are done with MS Excel).
Solutions
·
Fixing
solution: 50 % ethanol, 10 % glacial acetic acid, ad 100 % with aqua dest.
·
Incubating
solution (1L): 30 % ethanol, sodiumthiosulfate anhydrous 2g, sodiumacetat anhydrous 34 g, fill up to 1L
with aqua dest. Before use add 125 µL of glutaraldehyde/50 mL incubating
solution.
·
Silvernitrate
solution (1L): AgNO3 1 g, dissolved in 1L aqua dest.. Before use add 10 µL of formaldehyde/50 mL of silver
nitrate solution.
·
Developing
solution (1L): Na2CO3 anhydrous 25 g, dissolved in 1L
aqua dest.. Before use add 10 µL of formaldehyde/50 mL of developing solution.
·
Stop
solution (1L): sodium-EDTA 15.78 g dissolved in 1L aqua dest..
After electrophoresis, the polyacrylamide gel is taken
out of the casting sandwich and placed in a clean glass beaker filled with
fixing solution. All following steps are
carried out while gently shaking. The gel has to be incubated with the fixing
solution for 30 minutes. After fixation an appropriate amount of incubating
solution including glutaraldehyde (the gel has to be at least covered by
liquid) is prepared and added to the gel, followed by incubation for 15
minutes, discarding the fixing solution and washing with aqua dest. 3x for 5
minutes and 10 minutes incubation in silvernitrate solution including formaldehyde.
The silvernitrate solution is collected (special waste). Developing is carried
out by incubating the gel in developing solution including formaldehyde until
the desired intensity of protein staining is reached, followed by discarding of
developing solution and adding stop solution. The gel should incubate for at
least 1 hour in the stop solution. Afterwards the gel can be stored in aqua
dest. or dried with vacuum.
Solutions
For reprobing PVDF membranes the previous antibody has
to be removed by a socalled stripping procedure. An appropriate amount of
stripping buffer (about 25 ml per blot) is prewarmed to 50°C, the membrane is
laid on the inner wall of a hybridization tube and together with the stripping
buffer it is incubated under rotation at 50°C in a hybridization oven for 30
minutes. After stripping, the membrane is washed twice for 10 min in PBS/0.5%
Tween 20 and blocked again (by incubation in 5 % milk powder in PBS-Tween). It
is then ready for the next immunoblotting.
Solutions:
A blue p1000 tip was plugged with a p200 filter, then
0.5 mL of asubunit a4 coupled to agarose beads were placed on the filter.
The generated small column is equilibrated with 3 x 1 mL proteasome wash buffer
by gravity flow. 293 or HeLa cells are lysed
with proteasome activity lysis buffer and precleared as described above under
“cell extract generation”. The cell extract is loaded on the column by gravity
flow, the column is washed with 3 x 1 mL physiological proteasome wash buffer.
Elution is performed with 3 x 1 mL high salt proteasome elution buffer. Elution
fractions are collected and investigated for proteasome activity with the
fluorogenic substrate Suc-LLVY-AMC. The proteasome fractions are either
dialysed or TCA precipitated. The column is reequilibrated with proteasome wash
buffer and stored in PBS def. containing NaN3 to prevent bacterial
growth.
Solutions:
Samples with salt concentrations of 2 M are
transferred to a reaction tube, the lid was removed and instead a dialysis
membrane (cut off) is fixed. This small dialysis chamber was fixed up side down
on the wall of a big glass beaker filled with about 300 mL dialysis buffer.
Dialysis was carried out o/n at 4°C.
Preparing: a glycerol gradient of 35 – 80% glycerol is
used. Glycerol dilutions from 35 - 80% are prepared in 5 % steps. Thick walled
centrifugation tubes are placed upright in a rack over night at –80°C. For
pouring the gradient, 120 µL of each glycerol dilution are pipetted in the
centrifugation tube starting with the heaviest (80% glycerol). After each
dilution the rack is placed back at –80°C for 2 minutes until the layer is
either very viscous or frozen. Then the next layer is pipetted on top and so
forth. With that procedure a very sharp gradient can be created. On top of the
last frozen layer the sample of 120 µL are pipetted. Then all tubes are
balanced on an analysis balance.
Centrifugation parameters (Beckmann Ultracentrifuge
Optima TLX): 40000 rpm, 4°C, vacuum, 16 hours. After centrifugation, the
gradient was fractionated manually with a Gilson p200 120 µL steps each.
Solutions:
Protocol (Glickman et al. 1998a)
slightly modified
The native PAGE gels containing 4 %
acrylamide/bisacrylamide are poured without a stack, for 2 gels (6 cm x 9 cm x
0.015 cm) 25 mL of separating gel are prepared as follows: 21.3 mL native separating
gel buffer and 3.7 mL acrylamide/bisacrylamide mix are mixed thoroughly
avoiding bubbles, 250 µL 10 % APS and 25 µL TEMED are added to start
polymerization. The solution is poured immediately into the prepared gel cast
cassette and the combs are inserted. After 30 minutes when polymerization is
finished, the gel sandwich is placed in the electrophoresis tank and running
buffer is poured in both electrode chambers. 20 µL samples are mixed with 5 µL
of loading dye and loaded on the gel. Electrophoresis parameters: 30 mA for 1
gel. The gel is running for about 2 hours until the blue front eluated into the
lower running buffer chamber. In most cases, the native gel is afterwards
subjected to a proteasome activity overlay assay.
Solutions
10 µL of cell extract are mixed with 90 µL proteasome
activity buffer and 1 µL of Suc-LLVY-AMC solution and vortexed well. With a 1
mL syringe the sample is injected bubblefree into the fluorescence detector
(model: FP-920 Jasco Inc, Japan). The measuring parameters of the acquisition
method are: Time scan for 5 min at an excitation wavelength of 380 nm and
emission wavelength of 440 nm with data acquisition every 5 seconds. The gain
is set to 10 (2nd stage of 4 logarithmic amplification steps) and
the emission bandwidth is set to 18 nm slit. Due to proteasome activity, the
fluorescent molecule AMC (7-amino-4-methyl coumarin) is released from the
non-fluorescent peptide substrate Suc-LLVY-AMC resulting in a linear increase
of fluorescence over time with the slope being proportional to proteasome
activity. The resulting graph of proteasome activity is then exported to MS
Excel and evaluated. The slope of the fluorescence increase is calculated by
linear regression and normalized by total protein amount to determine the
normalized proteasome activity. It has to be noted that the fluorogenic peptide
substrate is cleaved by proteasomes independent from ubiquitination and most
likely also independent from the proteasome activator complex.
Solutions:
After electrophoresis, the native 4 % PAGE gel is
carefully taken out of the gel sandwich and placed on a plastic wrap. About 5
mL of overlay solution containing 5 mL proteasome activity buffer and 50 µL of
Suc-LLVY-AMC solution arere spread evenly over the surface of the native gel
and the gel is soaked for about 10 minutes. Fluorogenic peptide activity is
detected with the Sybr green filter of the LumiImager™ (Roche) detection
system.
Solutions:
The samples are mixed with trichloro acetic acid
solution (TCA) to a final concentration of 10 % w/w. After incubation for 1
hour at 4°C the samples are centrifuged for 30 minutes at 17.000 x g at 4°C in
a tabletop centrifuge. The supernatant is removed and the pellet is washed once
with cold acetone – followed by drying for 5 min and uptake in the appropriate
buffer (e.g. SDS-PAGE buffer).
Solutions
·
Kinase
lysis buffer: Hepes 10 mM pH 7.9, b-Glycerophosphate 20 mM, Na-Orthovanadate 1 mM, NaF 1
mM, DTT (dithiothreitol) 1 mM, Na-EDTA 2 mM, NaCl 150 mM, 1 % Triton X 100,
rest of volume aqua dest.
·
Kinase
buffer: Hepes 10 mM pH 7.9, b-glycerophosphate 12.5 mM, MgCl2 5 mM, NaF
1 mM, DTT 1 mM, ATP 50 µM pH 7.4, NaCl 50 mM, rest of volume aqua dest. Add freshly before use: MnCl2 1 mM
·
32P g-ATP 10 µCi/µL (Amersham)
Immunoprecipitation of flag IKK2wt with M2 agarose
beads is carried out as described above. The beads are washed 2x with cold PBS
and 2x with kinase buffer and the kinase buffer is quantitatively removed by a
capillary Pasteur pipette or a thin electrophoresis application pipette tip.
Per sample 10 µL kinase buffer and 25 µCi 32P g-ATP (2.5 µL) are mixed and preincubated for 10
minutes at 37°C. 12.5 µL of this preincubated kinase buffer mix and 8 µL COP9
substrate or 1 µL IkBa substrate are pipetted on the wall of the reaction
tubes containing the immunoprecipitation sample. The solutions are briefly
centrifuged and mixed quickly in order to avoid contact between kinase and
substrate without 32P g-ATP. The kinase reaction is
carried out for 2 hours at 37°C and is stopped by adding 6x protein sample
buffer. Then the kinase samples are subjected to 12.5 % SDS PAGE, silver
staining is performed, the silver-stained gel is vacuum-dried and exposed to
phosphor screens or Kodak X-OMAT films.
Fixed and silver-stained SDS-PAGE gels are sandwiched
between a layer of Whatman filter paper and plastic wrap, the sandwich is
placed in the vacuum dryer with the wrap layer facing up. The gel is dried for
about 15 minutes at 80°C with vacuum, and then the dried gel is cooled down
between two heavy objects to decrease deformation and cracking. The dried gel
covered with a plastic wrap is exposed to a phosphor screen o/n or to Kodak
films for some days depending on signal intensity. The phosphor screens are
scanned with Storm™ equipment (Molecular Dynamics).
.
The green
fluorescent protein (GFP) was discovered in the jellyfish Aequorea victoria
(figure 3.2) as a protein fluorescing without the need for any substrates or
coenzymes. The organism A. victoria contains both GFP and the chemiluminescent
protein aequorin. Upon light emission by aequorin GFP absorbs this light and
fluoresces green. As soon as the GFP gene was cloned and successfully expressed
in non-jellyfish organisms the way was clear for GFP to use it as tracking tool
fused to various proteins of interests.
Fluorescence is a phenomenon of molecules that absorb
light of a certain wavelength and emit light of a longer wavelength. Absorption
of light is also called excitation, whereas this energy is not kept within the
molecule but is emitted again. Emission wavelengths are always redshifted by
that means nearer to the red spectrum and a longer wavelength than the
excitation wavelength. Figure 3.3 shows an example of excitation and emission
spectra.
GFP has a molecular weight of 28 kDa. Crystal
structure data of GFP dimers reveals a barrel like structure composed of b-sheets designated as b-can structure. (figure 3.4) However, some biochemical
properties of wt GFP such as folding, dimerization, absorbance peaks,
fluorophore formation and expression levels were not satisfying. Therefore
amino acid mutations were generated in order to improve the properties of
wildtype GFP (wtGFP). The evolved GFP with “special features” was then named
enhanced GFP (EGFP). EGFP has a much faster fluorophore formation, 0.45 hours
instead of 2 hours and it produces a much brighter and stable GFP (mutation Ser65
ŕThr65 about 35-fold brighter) and it could
be expressed correctly folded at 37°C. Protein dimerization at high expression
levels could be diminished with mutations like Ala206 ŕ Lys206, Leu221ŕ Lys221 or Phe223 ŕ Arg223. Wild type GFP possesses two
absorbance peaks which are reduced to one in EGFP to increase specificity of
excitation (mutation Ser65 ŕThr65, Ala65, Gly65, Cys65
or Leu65). WtGFP’s temperature sensitivity could be removed by
introduction of the mutation Phe64 ŕ Leu64.
The use of EGFP as tracking molecule
offers the possibility to study living systems with a non-destroying approach,
given the fact that EGFP does not interfere with the properties of the protein
of interest. Certainly it has to be tested whether the EGFP-fusion protein
behaves like the untagged one, but in most cases EGFP is only little interfering
if at all.
EGFP fusion proteins are generated by
simply cloning the GFP gene up- or downstream of the gene of interest in an
appropriate DNA construct. To investigate how the GFP-fusion protein is
behaving in living cells, transfections are carried out and 24 hours after
transfection cells can be investigated by fluorescence microscopy, flow
analysis or other methods based on fluorescence. Comparisons of GFP-chimeric
proteins with wild type, untagged proteins can be done with reporter gene
assays, immunofluorescence as compared to GFP-fluorescence or other appropriate
biological tests. In addition to the development of enhanced, improved GFP
variants, other fluorescent protein versions were generated that differ from
GFP in the color of the emitted light (and also the excitation wavelength)
Blue (Tyr66 ŕ His66), cyan and yellow fluorescent
mutants with different absorbance and emission profiles were obtained (see
figure 3.5). This opened the possibility to track two (or in special
applications even more) fusion proteins with different GFP variants
simultaneously in one cell.
Confocal
microscopy differs from conventional light microscopy in the light source, the
detection, generation and resolution of acquired images. In a conventional
fluorescence microscope the whole specimen is illuminated by a certain
excitation wavelength, likewise emitted light is gathered from all planes of
the specimen. Visible light cannot be focused in a single plane, therefore not
only emitted light from the focal plane but below and above the specimen are
detected. This additional light detected results in a blurry image of decreased
contrast and resolution, especially for thick specimens. In contrast to
conventional light microscopy, in confocal microscopy a defined spot in the
focal plane of the specimen is illuminated at a certain time point. Laser light
sources of defined excitation wavelengths are used. The laser light is focused
by passing through a very small aperture, such as a pinhole or a slit.
Furthermore, emitted light from below or above the focal plane is eliminated by
preventing passing a second pinhole (see figure 3.6, orange lines). By changing
the z-axis distance between specimen and objective lense, the focal plane can
be adjusted and so called optical slices of the specimen in X-Y plane can be
generated. This technique is a non-invasive approach to investigate both fixed
and living cells. Emitted light of an illuminated spot in the focal plane is
detected by photomultiplier tubes (PMT), which enhance signal. Series of
illuminated PMT outputs are processed to an image.
Solutions:
293 or HeLa cells are passaged and seeded at a density
of 500.000 cells/6-well (40 % confluence), each 6-well contains a round sterile
glass cover slip. 1 day after lipofectamine or CaCl2 transfection of
293 or HeLa cells with fluorescent fusion proteins, the culture medium is
removed, the cells are washed once with PBS def. and about 2 mL 4 %
paraformaldehyde solution per 6-well are carefully pipetted to the cells. After
about 10 minutes incubation at room temperature the paraformaldehyde solution
is removed. The fixed cells are washed with PBS and covered with mounting
medium. Then the coverslip is carefully taken out of the 6-well and placed on a
glass slide with the cells facing the slide, trying to avoid bubbles. Excess of
mounting medium is removed and clear nailpolish is applied on the edges of the
coverslip to seal the fixed sample to prevent drying. Fluorescent microscopy is
then performed with the sample.
293 or HeLa
cells are seeded in 6-well plates containing round sterile coverslips
(diameter: 15 mm), the next day lipofectamine or CaCl2 transfection
is carried out. 1 day after transfection the round coverslips are carefully
taken out of the well and pressed on the hole of an aluminum slide (figure
1.7). The edges of the aluminum slides are greased to seal the cover slips.
About 70 µL of DMEM complete medium is pipetted on the cells on the coverslip.
With a second blank coverslip pressed on the other side of the aluminum slide a
chamber filled with medium is created. The aluminum slide is placed on the
object table with the cells grown on the coverslip on the bottom.
The FRAP
technique allows to investigate molecular dynamics of fluorescent proteins like
EGFP in a living cell being a non invasive microscopy technique. This
microscopy technique consists of 4 steps: a pre-bleach scanning of the whole
cell, bleaching of a pre-defined region with 100 % laser intensity, a time
series scanning and a post bleach scanning image. The pre and post bleach
images are later used for calculation of corrected FRAP. During a bleaching
process laser light of high intensity irreversibly destroys the fluorophore and
significantly decreases the mean fluorescence in the defined bleach region.
Then a set of scan images are taken to observe the increase of fluorescence
(recovery) due to diffusion processes in and out of the scan region (FRAP
scheme see figure 3.9). The diffusion velocity of the fluorescent protein is
indirect proportional to its molecular weight. The recovery behavior of a
fluorescent protein after a bleaching process is typical for its cellular property.
Proteins can diffuse rather undisturbed or are incorportated in larger
complexes and structures.
FRAP gives
information about relative amounts of mobile and immobile fractions of the
fluorescent protein abundant in the cell. First an
image of the whole cell is captured before bleaching. Both, a round scan and
bleaching area in the cell are defined. The following acquisition of time
series are performed: 1 image before bleaching, 70 iterations of bleaching with
100 % laser power, 100 scans of the bleach area with averaging of 4 scans are
taken. An image of the whole cell is captured after the bleaching and recovery
process. The image series are exported to TIF files with LSM Image Examiner and
later evaluated with Scion Image. The bleach region in each image of the time
series is measured for its mean fluorescence using the “measure all” command of
the Measure Macro of Scion Image. Resulting mean fluorescence data is
transferred and evaluated in MS Excel and Graphpad Prism. Fluorescence recovery
data have to be corrected due to co-bleaching of the whole cell, by that means
the total cell mean fluorescence decreases and therefore a recovery of e.g. 100
% would never be achieved even though the fluorophores are absolutely mobile.
The FRAP correction factor is obtained by the ratio of total cell fluorescence
Fpost/Fpre = Fcorr. All fluorescence recovery
intensities (except for prebleach scan) have to be divided by this correction
factor. The recovery period could be modelled best with a one phase exponential
equation:
Y [%fluorescence)= Span * (1- e1-kx) +
Bottom
This curve starts at the bottom and increases to
bottom + span (= plateau) with a rate constant K. The half life is calculated
with t1/2= ln2/K or t1/2= 0.693/K.