Hi Sakshi

Please find the protocol for protein extraction for western blot below.

https://www.abcam.com/protocols/sample-preparation-for-western-blot

Also, find attached the bradford assay protocol below.

https://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html

Best Wishes.

collect the cell lines via centrifugation then add RIPA lysate accordingly and then Perform ultrasonic sonication and then centrifuge to collect the protein. after that check the concentration via following the instruction of BCA kit. Calculate the concentration according to C1V1=C2V2, add 5×loading buffer, mix and cook for 5-10 min in 100°C metal bath.

I would say RIPA buffer is not compatible with Bradford assay in 96-well plate format for protein determinatons. Use BCA reagent instead.

I use 30 min at 4⁰C as a general rule for lysis (for whatever detergent used in the lysis buffer), but witt RIPA you will have nuclear disruption , so beggining minute 15 you have to start dong vórtex and lot of pipetting to shear DNA (if you don't have a sonicator). If too viscous you may need to pass the lysate through a 1 mL syringe (very high gauge, i.e. small claliber). Finally, if viscosity is ok (pipettable) you can centrifuge to pellet cell debris (at 4⁰C). Always keep temperature 4⁰C at every single step, starting PBS wash if working with adherent cells in tissue culture.

Good luck!

https://www.researchgate.net/deref/https%3A%2F%2Fwww.abcam.com%2Fprotocols%2Fsample-preparation-for-western-blot