Western blot | Bradford assay ⎸Protein quantification

Lab. Bioclock & Aging/Dewan Md. Sumsuzzman

Bradford assay ⎸Protein quantification

A. Preparation of BSA standard curve

Protocol:

1. Preparation of 10 mg/ml BSA solution (10 ml DW + 100 mg BSA) [Stock solution]
2. Prepare 1 mg/ml BSA solution (9 ml DW + 1 ml of 10 mg/ml BSA) [Working solution]
3. Levelling total 8 cuvettes as Blank, 1, 2, 4, 6, 8, 10, 12 µL respectively.
4. For blank, add 1 mL lysis buffer. And, add 1 mL working solution to 1, 2, 4, 6, 8, 10, 12 µL levelled cuvettes respectively.
5. Discard 1, 2, 4, 6, 8, 10, 12 µL working solution from 1, 2, 4, 6, 8, 10, 12 µL levelled cuvettes gradually.
6. Start quantification and get a BSA curve.

B. Protein quantification

Protocol:

Part-1 (Prepare working solution):

1. Prepare lysis buffer (Western blot protocol)
2. Mix the tissue with lysis buffer in a conical tube (10 mg tissue/1 mL lysis buffer) and incubate for one hour.
3. Prepare bradford working solution by mixing bradford stock solution and autoclaved DW with a ratio of 1:4 [{Dilution formula, M1V1 = M2V2}; {200µL ✖ 1mL = 800µL ✖ 1mL}] [Working solution]
4. Centrifuge at 13000 rpm in 4℃ for 20 mins
5. Transfer the supernatant to new e-tube

Part-2 (Start quantification):

1. Aliquot 1 mL (100 µL) of bradford working solution.
2. Discard 1 µL of  bradford working solution
3. Add 1 µL of sample [that you want to quantify]
4. Gently pipetting and incubate for 5 mins
5. Set the wavelength of spectrophotometer 595 nm
6. Quantification.