Protein Methods

• Cytosolic Cell Extract Preparation

• Protein Quantification (Bradford)

• Immunoprecipitation

• Co-Immunoprecipitation to detect Protein Interactions

• Denaturing SDS-PAGE

• Electro Transfer Blotting on PVDF Membrane

• Western Blot

• Silver Staining of PAGE Gels

• Stripping of PVDF Membranes

• Proteasome Purification by Immunoprecipitation

• Proteasome Activity Assay

• Proteasome Activity Overlay Assay

• Dialysis

• Glycerol Gradient Centrifugation

• Native PAGE

• Trichloro-Acetic Acid Precipitation of Proteins

• Kinase Assays in vitro

Cytosolic Cell Extract Preparation

Solutions:

• Nonidet P-40 (NP 40)

• Protease inhibitor cocktail (1 tablet Complete™ protease inhibitor, Roche) in 1 mL PBS makes 50x stock solution): pepstatin, aprotinin, leupeptin, phosphoramidon

• Lysis buffer: PBS def. containing 0.5 % (v/v) NP 40 and protease inhibitors (when the protein concentration had to be determined by Coomassie staining the NP-40 concentration was decreased to 0.2% to reduce interference with the protein quantification).

• Proteasome activity lysis buffer: Tris/HCl 10 mM pH 7.4, 10 % (v/v) glycerol, KCl 150 mM, MgCl₂ 10 mM, ATP 5 mM (freshly added prior to use), 0.2 % NP 40

• Adenosintriphosphate (ATP) 100 mM pH 7.4 in PBS complete (PBS deficient containing additionally CaCl₂ 1mM, MgCl₂ 0.5 mM

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.

Protein Quantification (Bradford)

Solutions:

• BSA 1 µg/µL in PBS

• BioRad 1:5 Coomassie Reagent diluted in AD

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.

Immunoprecipitation

Solutions:

• 1 % BSA in PBS

• PBS def., 4°C

• Agarose beads: aflag agarose beads (M2-aflag affinity matrix, Sigma, Cat #A-1012), aIKK1/2 agarose beads (Santa Cruz)

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).

Co-Immunoprecipitation for the Detection of Protein Interactions

 1.     Transfection of cells with tagged proteins (one 6-well of CHO or HeLa cells is sufficient for one sample).

2.     Preparation of extracts:

2.1.   1 d after transfection: wash cells with PBS

2.2.   Lysis with 500 µl/well Lysis-Buffer + Protease Inhibitors: 15 min at 4°C.
Buffer: 0.5% NP40, 50 mM Tris/HCl pH 7.5, 1 mM EDTA, 150 mM NaCl.
Protease Inhibitors: 10 µg/ml Aprotinin, 20 µg/ml Phosphoramidon, 40 µg/ml Pefabloc, 1 µg/ml Leupeptin, 1 µg/ml Pepstatin (from 1000x stock solutions, Boehringer Protease Inhibitor set).The lysis is suited for cytosolic proteins and membrane proteins. Nuclei remain intact (you can leave the nuclei on the plate when you take off the supernatant).

2.3.   Spin the extracts for 15 – 30 min at 14 krpm, 4°C (HeLas: 15 min, CHO: 30 min)

2.4.   Keep the supernatant and adjust the NaCl-concentration (150 mM – 1000 mM depending on the strength of interaction; start in the range of 150 – 250 mM, increase the concentration if you want to increase the stringency)

3.     Co-Immunoprecipitation

3.1.   Take 400 µl of extract for IP (keep about 30 µl extract for direct western analysis).
Use flat-top tubes (the visibility of the pellet is better in these tubes) Add 400 µl Lysis-Buffer/250 mM NaCl (without NP40 > final concentration: 0.25%). Add beads (15 µl anti-flag-M2-Agarose, Sigma A-1205; alternatives: other antibodies directly coupled to CNBr-activated Sepharose; Protein A- or Protein G-Agarose: the later will give more unspecific binding). Rotate extracts + beads for 2 h at 4°C.

3.2.   Spin for 30 sec at 14 krpm 4°C. Take off the supernatant, add 1 ml of lysis buffer/250 mM NaCl/without NP40 and invert tubes several times (do not vortex). Repeat this washing step.

3.3.   Suspend the beads in 1 ml cold PBS and transfer the suspension to a new tube.
Spin 30 sec at 14 krpm, 4°C, take off the supernatant and repeat this washing step. Final centrifugation: 1 min at 14 krpm, 4°C. Remove the supernatant and suspend the beads in SDS-PAGE buffer (30 µl). Incubate for 5 min at 95°C and pellet the beads for 2 min at 14 krpm.

4.     SDS-PAGE

5.     Western Blot: if possible use HRP-conjugated primary antibodies (anti-HA-HRP from Boehringer, anti-myc-HRP from Invitrogen). This gives much lower background of unspecific bands (Ig light chain …).

Denaturing SDS-PAGE

Solutions:

• Solution A: 1.25 M Tris/HCl pH 8.8, 0.4 % SDS

• Solution B: 30 % Acrylamide/N’, N’-Bismethyleneacrylamide (29:1) in H₂O

• Solution A Stack (ASt): 0.5 M Tris/HCl pH 6.8, 0.4 % SDS, phenol red

• Ammoniumpersulfate (APS) 10 % (w/v) in AD

• N, N, N’, N’- Tetra-ethylethylene-diamine (TEMED)

• Butanol

• 1x SDS running buffer: Tris base 25 mM, glycine 250 mM, 0.1 %SDS

• 6x protein loading dye: Tris/HCl 750 mM pH 6.8, 60 mM DTT, 12 % SDS, 60 % glycerol anhydrous, bromophenol blue

• prestained protein marker: Benchmark™ (INVITROGEN)

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.

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.

 Electro Transfer Blotting on PVDF Membrane

Solutions

• 1x SDS western transfer buffer (1L): 25 mM glycine (2.9 g), 50 mM Tris base (5.8 g), 20 % methanol (200 mL), 0.4 % SDS, aqua dest. ad 1L, pH should be 8.0

• Methanol 100 %

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.

 Western Blot

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

·         Wash solution: 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, Santa Cruz), aflag tag M2 (1:2000, mouse, Sigma), aNEMO/IKKg (1:500, rabbit, Santa Cruz), a HA tag (1:1000), aIKK1 (1:500, Santa Cruz)

·         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).

Silver Staining of PAGE Gels

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.

Stripping of PVDF Membranes

Solutions

• Stripping buffer: 62.5 mM Tris/HCl pH 7.0, 2 % (w/w) SDS, 0.7 % mercaptoethanol

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.

Proteasome Purification by Immunoprecipitation

Solutions:

• Proteasome activity lysis buffer: Tris/HCl 10 mM pH 7.4, 10 % (v/v) glycerol, KCl 150 mM, MgCl₂ 10 mM, ATP 5 mM pH 7.4 (freshly added prior to use), 0.2 % NP 40

• Proteasome wash buffer: Tris/HCl 10 mM pH 7.4, 10 % (v/v) glycerol, KCl 150 mM, MgCl₂ 10 mM, ATP 5 mM (freshly added prior to use)

• Proteasome elution buffer: Tris/HCl 10 mM pH 7.4, 10 % (v/v) glycerol, KCl 2 M, MgCl₂ 10 mM, ATP 5 mM pH 7.4 (freshly added prior to use)

• a20S beads: antibody against subunit a4 coupled to agarose beads (Affinity Ltd, UK, PW 9005, Batch Z04562)

• NaN₃ 10 mM in PBS def.

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.

Proteasome Activity Assay

Solutions

• Fluorogenic peptide solution: Suc-Leu-Leu-Val-Tyr-AMC (Suc-LLVY-AMC) 1 mg/mL in N,N –Dimethylformamide (100x)

• Proteasome activity buffer: Tris/HCl 10 mM pH 7.4, KCl 140 mM, MgCl₂ 5 mM, ATP 5 mM

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 (2^nd 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.

Proteasome Activity Overlay Assay

Solutions:

• Fluorogenic peptide solution: Suc-Leu-Leu-Val-Tyr-AMC (Suc-LLVY-AMC) 1 mg/mL in N,N –Dimethylfomamide (100x)

• Proteasome activity buffer: Tris/HCl 10 mM pH 7.4, KCl 140 mM, MgCl₂ 5 mM, ATP 5 mM

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.

Dialysis

Solutions:

• Dialysis buffer: Tris/HCl 10 mM pH 7.4, 10 % (v/v) glycerol, NaCl 100 mM, MgCl₂ 10 mM, ATP 5 mM pH 7.4 (freshly added prior to use)

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. 

Glycerol Gradient Centrifugation

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.

Native PAGE

Solutions:

• Native separating gel buffer / running buffer: 0.18 M Tris/borate pH 8.3, MgCl₂ 5 mM, DTT 1 mM, 1 mM ATP

• Acrylamide/N’, N’-bismethyleneacrylamide mix: 37.5:1

• Ammoniumpersulfate (APS) 10% w/w

• N, N, N’, N’- Tetra-ethylmethylen-diamine (TEMED)

• Adenosintriphosphate (ATP) 100 mM pH 7.4 in PBS complete

• Native loading dye: 50 % glycerol containing 0.0025 % xylene cyanol

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.

 Trichloro-Acetic Acid Precipitation of Proteins

Solutions:

• TCA 50 % w/w

• Acetone

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).

Kinase Assays in vitro

Lysis buffer (final conc.):          for 20 ml:

Protease Inhibitors: added before use (Leupeptin, Pepstatin, Pefa-Block) according to stock

Kinase buffer (final conc.):                   for 20 ml:

1. Lyse cells (in 6 wells) with 500 µl per well of Lysis buffer (+ protease inhibitors):
   20 min at 4°C.

2. Clear by centrifugation (14000 rpm, 4°C 15 min Eppendorf centrifuge). Save an aliquot (30 µl) for Western blotting.

3. Immunoprecipitate the kinase (e.g. with 10 µl anti-flag affinity matrix beads, Sigma, for flag-tagged transfected kinase; or with appropriate antibody for endogenous kinase + Protein A-Sepharose or directly coupled to agarose): 2h at 4°C (rotating).

4. Wash the beads: 3x with 1 ml PBS (4°C), 1x with 1 ml Kinase buffer (4°C): pellet the beads by centrifugation (14000 rpm, 4°C, 45sec) and remove the supernatant.

5. Prepare Kinase buffer: add MnCl₂ to 10 mM (stock: 1 M) and ³²P-γ-ATP (5 µCi per sample, usually 1/10 volume, i.e. 1 µl of stock solution for one 10 µl assay) and preincubate at 30°C for 10 min.

6. Add  1 µg substrate: GST-IκB (1 µl) or mutant substrate (as control) to the beads; add 10 µl kinase buffer, mix gently and incubate at 30°C for 30 min (or longer).

7. Stop the reaction by addition of 4x SDS-sample buffer (4 µl) and perform SDS-PAGE with the samples, followed by fixation of the gel (10% methanol, 10% HAc), drying and autoradiography.

For detection with PhastGel: use only 5 µl beads, 5 µl kinase buffer, 0.5 µl substrate and 2 µl 4x SDS-sample buffer: Run a 12.5% PhastGel with 4 µl per sample

Detection of ³²P g-ATP Phosphorylated Proteins

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).