Western Blot Analysis from kidney wedge biopsies

Judith Sunzenauer (last update 1.4.2015)


Sample Preparation:

  • Cool centrifuge to 4°C
  • Heat thermoblock to 95°C
  • Dissolve 1 tablet of Protease Inhibitor (Complete Tablets Mini EASYpack, Roche) in 1ml RIPA buffer (store at 4°C for a 1 week or at -20°C for long term storage)
  • For each kidney wedge biopsy add 100µl of Protease Inhibitor Solution to 900µl RIPA buffer (50mM Tris-HCl pH 8.0, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1mM NaF, 1mM sodium orthovanadate). Keep on ice
  • Merge the tissue using a autoclaved tissue grinder
  • Sonicate the tissue lysate for 15sec to complete cell lysis and shear DNA to reduce sample viscosity
  • Spin at 16,000g for 20min in precooled centrifuge
  • Transfer the supernatant to a fresh tube. Keep samples on ice
  • If necessary, aliquot the protein samples for long term storage at -20°C
  • Prepare appropriate amount of 2x Laemmli sample buffer by adding 50µl β-Mercaptoethanol to 950µl of 2x Laemmli sample buffer
  • Take 20µg of each sample and add an equal volume of 2x Laemmli sample buffer
  • Boil each cell lysate in sample buffer at 95°C for 5min
  • Centrifuge at 16,000g for 1min.


  • Take a Mini-PROTEAN TGX gel, remove the comb and the tape from the bottom of the cassette
  • Place the cassette in a BioRad Mini-Protean Tetra System chamber and fill each integrated upper buffer chamber with running buffer (25mM Tris, 190 mM glycine, 0.1% SDS, pH8.3)
  • Fill the lower buffer tank with running buffer to the marked fill line
  • Load 20µl of the protein samples and 5µl of protein marker
  • Place the lid on the tank, aligning the color-coded plugs with corresponding jacks on the lid
  • Run the gel for 5min at 50V
  • Increase the voltage to 200V to finish the run in about 20-30min.


  • After electrophoresis is complete, turn off the power supply and disconnect the electrical leads
  • Remove the gel cassette from the cell. Pull the two plates of the cassette apart to expose the gel
  • Open a Trans-Blot Turbo Mini PVDF transfer pack and place the pad with the membrane on the base of the transfer cassette
  • Carefully lift the gel from the cassette and place it on top of the membrane, place the top pad on the gel and roll out bubbles
  • Place the lid on the cassette base and lock it
  • Insert the cassette into either instrument bay. Start the transfer by selecting preset Turbo program and choosing the appropriate gel size and press RUN.
  • A typical run takes 7min
  • When the transfer is over, disassemble the blotting sandwich and place both the blot and the gel in a container with deionized water.

Visualize with Ponceau Red:

  • Remove the blotting membrane from the container and place in Ponceau S Solution for a few seconds
  • Cut membrane if necessary
  • Rinse the blot 2 times for 5min in a.dest.

Antibody incubation:

  • Rinse the blot for 5min in TBST (0,1% Tween added to 1x TBS containing 20mM Tris, 0.9% NaCl, pH 7.4)
  • Block in 3% BSA in TBST at room temperature for 1h
  • Incubate overnight in the primary antibody solution at 4°C. Use antibody dilutions in blocking buffer according to manufacturer´s recommendations as listed below:
Product ID Protein Size Dilution
ab154163 Klotho 150 kDa 1:1000
ab52857 Foxo1 70-80 kDa 1:1000
ab59348 BCL2 26 kDa 1:1000
ab9485 GAPDH 37 kDa 1:2500
ab156302 β-Actin 42kDa 1:3000
  • Rinse the blot 5 times for 5min with TBST
  • Incubate in the HRP conjugated secondary antibody solution (goat anti-rabbit 1:3000) for 1h at room temperature
  • Rinse the blot 5 times for 5min with TBST.

Imaging and analysis using the MicroChemi (DNR Bio-imaging systems) System and GelQuant Software:

  • Prepare Clarity western ECL substrate mixture in a 1:1 ratio. Prepare 0.1ml of solution/cm2 of blotting membrane
  • Incubate membrane with approximately 2ml for 5min
  • Place the blotting membrane on the sample stage of the MicroChemi and start AUTODETECT
  • Save as .tif
  • Export as .wmf
  • Open GelQunat Software and select “1D Gel or Western Blot analysis”
  • Open .tif file
  • Use “automatic” or “stepwise” calculation of bands
  • Select measurement window and select export as file
  • Save .txt file.


Complete Tablets, Mini EASYpack Roche 04693124001
RIPA Buffer Sigma Aldrich R0278-500ml
PYREX Tenbroeck Tissue Grinder VWR 89090-892
2x Laemmli Buffer BioRad 1610737
Trans-Blot Turbo Transfer Pack BioRad 170-4156
Mini-PROTEAN TGX Gels BioRad 456-1093
10x Tris/Glycine/SDS Buffer BioRad 161-0732
Ponceau S Solution Sigma Aldrich P7170-1L
10x Tris Buffered Saline Sigma Aldrich T5912-1L
Blocker BSA in TBS (10x) Thermo Scientific 37520
Precision Plus Protein WesternC Standards BioRad 161-0376
Goat anti-rabbit HRP abcam ab6721
Clarity western ECL BioRad 170-5061