Western blot in homogenised mouse brain samples
Stephanie J Cragg, Katherine Brimblecombe, Natalie Connor-Robson
Abstract
This protocol is for western blot analysis of proteins in homogenised mouse brain samples. The sensitivity and selectively of this assay is dependent on the efficacy of antibodies. When using novel antibodies ensure they have been validated for western blot, ideally using tissue from knock-out animals.
Steps
Preparing Samples
Take samples out of the -80°C freezer and keep on dry ice until ready to digest.
On wet ice, add 200 µL RIPA Buffer (see Materials ) to each unilateral striatum sample.
Mix thoroughly until sample completely blended with Tissue Tearor.
Centrifuge samples at 6g for 5 min at 4ºC.
Using the supernatant, dilute samples to about 1:10 to determine protein concentration.
Analysing Protein Concentration
Prepare pre-diluted/standards: 1.0, 0.8, 0.6, 0.4, 0.2 and 0.0 mg/mL of Bovine Serum Albumin (BSA)
Prepare solution A:
- 320 µL Copper (II) Sulfate Solution
- 16 mL Bicinchoninic Acid Solution
In a well plate, add 10 µL of pre-diluted samples/standards + 100 µL solution A.
Do this in triplicate.
Cover plates with film and incubate the samples for 30 min at 37ºC.
Measure the absorbance of each sample at 562 nm using a spectrophotometer and create a standard curve to determine concentrations.
Running Western Electrophoresis Gel
Make up 83 µL samples in RIPA Buffer at a concentration of 20 µg/10 µL and then add 17 µL of 6x Loading Buffer (see Materials ).
Boil samples at this stage at 95 ºC for 5 min.
Prepare 1X Running Buffer (see Materials ) and add the pre-cast gel cassettes (4 – 12%).
Load 5 µL of sample per well.
Load visual + developable ladder in first lane, and only developable ladder in last lane.
Run electrophoresis for 60 min @ 200V/100mA.
Transfer gel onto membrane and transfer using machine.
Running Antibodies
Wash membranes with TBST (see Materials ) for approximately 1 min and repeat 4x.
Make up 50 mL TBST solution with 4% milk (dried powder).
Roll membranes into 50 mL conical with 3 mL of TBST w/ Milk and Primary Antibody.
The following primary antibody (working concentration 1:1000) was used in Brimblecombe, K. et al. (2023): anti-calb1 (Cell Signalling #13176).
Incubate overnight at 4 ºC or ~2 hours at room temperature.
Wash membranes with TBST for approximately 1 min and repeat 4x.
Incubate membrane in TBST w/ Milk and Secondary Antibody HRP conjugated (1:3000) for 1 hour at room temperature.
Wash membranes with TBST for approximately 1 min and repeat 4x.
Develop membranes with the chemiluminescent ECL Substrate Kit (~ 1 mL of each component).
Visualize gel on a Gel Doc System.
Put membrane back into TBST w/ Milk and the 1º/2º β-actin antibody (already conjugated, 1:50000).
Repeat steps 25 and 26 .
