Preparing sample for SDS PAGE

Question

I have more than 10 cell lysate samples (70 µL each) whose concentration varies from 1.9 mg/mL to 4.8 mg/mL. I have 5X and 2X SDS sample buffers. I would like to prepare SDS PAGE samples in such a way that the final concentration of all the protein samples would be equal. I am struggling to deal with it.

What I know is while using 5X buffer, for 4 parts of sample we use 1 part of buffer to make it finally 1X. Similarly, for 2X buffer, we use equal amounts of buffer and sample to make 1X. Also, I would like to know will it be a problem if I make it more than 1X? Why we need 1X?

Answer 1

Usually you would want to keep the amount same, not the concentration. However, if you still want the concentration to be the same then you can add suitable amounts of PBS or your lysis buffer.

For e.g. if you have two samples with 1mg/ml and 4mg/ml concentrations and you want to load 20μg of total protein, then you can take 20μl of first sample and add 5μl of 5x loading buffer. For the second sample, you would take 5μl of the sample, 5μl of 5x loading buffer and 15μl PBS.

Why 1x?

Because researchers before us found this composition to be optimal (or just simply stuck to it as a matter of practice).

Would it be a problem if the final concentration of the loading buffer is more than 1x?

Apparently not, in my experience if it is up to 1.5x. Never checked higher concentrations.

Answer 2

Your buffers come concentrated so that smaller volumes can be packaged, shipped and stored. They're intended for use at a 1X concentration, unless otherwise noted. So for example, if you get a 5X buffer then you should use an appropriate diluent to obtain a 1X concentration in the volume you need. This might be 5 mL (1 part) of 5X buffer into 20 mL (4 parts) of diluent to get 25mL of 1X buffer.

You would have to study the effect of using a more concentrated buffer in your process, but it can may affect your results, and it would certainly cost you more money.

A good question to pose yourself would be: If I use a more concentrated buffer for my SDS-PAGE, and there is variability in the results, how would I be able to tell if the variability came from the buffer concentration or not?

Instead, you can try bringing your protein samples up to the same concentrations first, and then dilute into your buffers as needed.