Judith Sunzenauer (last update 1.4.2015)

Procedure

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.

Electrophoresis:

• 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.

Blotting:

• 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:

• 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.

Material