Western blot for tissue extract
Veerle Baekelandt, María Sanchiz Calvo, Eduard Bentea
Abstract
Protocol for the detection of proteins by Western blot from tissue extract
Before start
Perform protein extraction from snap-frozen brain tissue:
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weigh tissue
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add RIPA buffer (see Materials) : 10 X of the weight (40 mg = 400 µL)
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homogenize samples using sample homogenizer
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sonicate samples at 4 degrees C, 3 times 15 seconds (keep the samples on ice between each sonication)
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centrifuge samples at 6000 g for 10 minutes at 4 degrees C
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collect supernatant and measure protein concentration
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aliquot protein extracts and store at -20 degrees
Steps
Day 1
Prepare sample for western blot
15µg of protein in 12µL
Add 4µL
Boil at 98°C for 0h 10m 0s
Load samples, together with 7µL of mass marker (PageRulerTM Plus Prestained Protein Ladder) on a on 4–15% CriterionTM Tris-HCl Protein Gel.
Run the gel for 0h 10m 0s at 80V
Run the gel for 0h 45m 0s at 150V
Transfer the proteins on PVDF membrane (Trans-Blot® TurboTM PVDF Membrane) using Trans-Blot Turbo Transfer System (Bio-Rad), using the pre-programmed protocol STANDARD SD: 0h 30m 0s , up to 1.0 A, 25 V.
Block membranes for 1h 0m 0s using 5% milk dissolved in PBS-T 0,1% at RTRoom temperature
Incubate membranes in primary antibody in 5% milk PBS-T 0,1% at 4°C
Day 2
Wash membranes with PBS-T 0,1% for 10 minutes, 3 times at TRoom temperature
Wash PBS-T 0h 10m 0s
Wash PBS-T 0h 10m 0s
Wash PBS-T 0h 10m 0s
Incubate membranes for 1h 0m 0s horseradish peroxidase-conjugated secondary
antibody (Dako) diluted 1/10000 in 5% milk PBS-T 0,1% atRoom temperature
Wash membranes with PBS-T 0,1% for 10 minutes, 3 times at TRoom temperature
Wash PBS-T 0h 10m 0s
Wash PBS-T 0h 10m 0s
Wash PBS-T 0h 10m 0s
Develop membranes using ECL Prime (GE Healthcare)
