If you are using 90 mm culture disks, your growth area is approximately 55 cm2. I think that 500 uL of RIPA buffer is an appropriate volume. I generally grow cells in 6 well plates with a growth area of 9.6 cm2 and lyse the cells in 100 uL. Your growth area is roughly 5 times larger than mine and you are using 5 times more buffer. I also obtain protein readings around 2 ug/uL using a BCA assay. You could certainly lower the amount of RIPA buffer to 400 uL.

What you need to ask yourself is how many Western blots are you going to perform. How many antibodies are you going to be studying. You can always add less RIPA buffer in order to get a more concentrated sample. You can definitely obtain higher concentrations around 5-7 ug/uL if you lysed your cells in lets say 250 uL. However, if you are running multiple western blots, you might run out of supernatant.

In order to load more protein into your gel, you can always use 4x or 6x sample buffer.

Thanks a lot for your answer, Brandon A Kemp .

I have noted what you suggested. I use 4x sample buffer.

These days, I reduce the lysis buffer amount to 300ul. However, one thing is still troubling me. That is; even if I tried to obtain the concentrated protein sample, let say 5ug/ul, can we still apply the BCA standard curve equation to calculate it?? (Because I am afraid that it is beyond the linearity range (up to 2mg/ml) according to the references.)

Yes, you can. However, when you perform your BCA assay on your samples, you need to dilute your samples in order for the protein reading to be within the linear standard curve. For instance, I take a small amount 5-10 uL of your sample in RIPA buffer and dilute it in pure water at 1:50. When you get your protein reading it should be within the range of the standard curve and then multiple by your dilution factor to get your final value. Does that make sense?

Yes, it makes sense now. Thank you so much for your teaching. It really helps me a lot. And so sorry for my late reply as I just noticed your answer.