TITLE: Western Blot
AUTHORS: Katherine Brimblecombe, Natalie Connor-Robson, Stephanie J Cragg

[DESCRIPTION]
TBD

[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.