TITLE: Western blot  in homogenised mouse brain samples
AUTHORS: Katherine Brimblecombe, Natalie Connor-Robson, Stephanie J Cragg

[DESCRIPTION]
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. 

[GUIDELINES]
Due to the samples being homogenised, biological replicates should be from individual animals. Technical replicates run on repeated samples from the same animal can be illustrative of the reliability of each reading, however, they do not reflect biological variability. 

[STEPS]
SECTION: Preparing Samples
1. Take samples out of the -80°C freezer and keep on dry ice until ready to digest. 
SECTION: Preparing Samples
2. On wet ice, add 200 µL RIPA Buffer (see Materials) to each unilateral striatum sample. 
SECTION: Preparing Samples
3. Mix thoroughly until sample completely blended with Tissue Tearor.
SECTION: Preparing Samples
4. Centrifuge samples at 6g for 5 min at 4ºC.
SECTION: Preparing Samples
5. Using the supernatant, dilute samples to about 1:10 to determine protein concentration. 
SECTION: Analysing Protein Concentration
6. Prepare pre-diluted/standards: 1.0, 0.8, 0.6, 0.4, 0.2 and 0.0 mg/mL of Bovine Serum Albumin (BSA)
SECTION: Analysing Protein Concentration
7. Prepare solution A: 
320 µL Copper (II) Sulfate Solution
16 mL Bicinchoninic Acid Solution
SECTION: Analysing Protein Concentration
8. In a well plate, add 10 µL of pre-diluted samples/standards + 100 µL solution A.

Do this in triplicate.
SECTION: Analysing Protein Concentration
9. Cover plates with film and incubate the samples for 30 min at 37ºC.
SECTION: Analysing Protein Concentration
10. Measure the absorbance of each sample at 562 nm using a spectrophotometer and create a standard curve to determine concentrations. 
SECTION: Running Western Electrophoresis Gel
11. 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).
SECTION: Running Western Electrophoresis Gel
12. Boil samples at this stage at 95 ºC for 5 min.
 

SECTION: Running Western Electrophoresis Gel
13. Prepare 1X Running Buffer (see Materials) and add the pre-cast gel cassettes (4 – 12%).
SECTION: Running Western Electrophoresis Gel
14. Load 5 µL of sample per well. 
SECTION: Running Western Electrophoresis Gel
15. Load visual + developable ladder in first lane, and only developable ladder in last lane.
SECTION: Running Western Electrophoresis Gel
16. Run electrophoresis for 60 min @ 200V/100mA.
SECTION: Running Western Electrophoresis Gel
17. Transfer gel onto membrane and transfer using machine.
SECTION: Running Antibodies
18. Wash membranes with TBST (see Materials) for approximately 1 min and repeat 4x.
SECTION: Running Antibodies
19. Make up 50 mL TBST solution with 4% milk (dried powder).
SECTION: Running Antibodies
20. 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).
 

SECTION: Running Antibodies
21. Incubate overnight at 4 ºC or ~2 hours at room temperature.
SECTION: Running Antibodies
22. Wash membranes with TBST for approximately 1 min and repeat 4x.
SECTION: Running Antibodies
23. Incubate membrane in TBST w/ Milk and Secondary Antibody HRP conjugated (1:3000) for 1 hour at room temperature.
 

SECTION: Running Antibodies
24. Wash membranes with TBST for approximately 1 min and repeat 4x.
SECTION: Running Antibodies
25. Develop membranes with the chemiluminescent ECL Substrate Kit (~ 1 mL of each component). 
SECTION: Running Antibodies
26. Visualize gel on a Gel Doc System. 
SECTION: Running Antibodies
27. Put membrane back into TBST w/ Milk and the 1º/2º β-actin antibody (already conjugated, 1:50000).
SECTION: Running Antibodies
28. Repeat steps 25 and 26.