The protein sample was injected into each well of the gel at pH2.5 without adjusting the pH value when I did the SDS-PAGE analysis. And finally, there is no brand. So, will the initial pH value of protein sample affect the results of SDS-PAGE analysis?
Thank you.
Dear Zhichao Lou
SDS is an anionic detergent which denatures proteins by eliminating their secondary and tertiary structures but also confers a negative charge to each protein nearly uniform in proportion to its mass along the length of the polypeptide chain. This allows separate proteins as a function of size. Therefore, in my opinion I do not think the pH you have altered the outcome of the SDS-PAGE analysis. I think it must have to do with the size of the protein so I suggest that you review the pore size or percentage of acrylamide you used.
Regards.
Dear Alain Bárbaro Alfonso Chaviano,
Thank you very much for your important suggestions.
Best.
Hi there,
I guess you have used a loading buffer to prepare your samples before loading them onto the gel? If so, as long as the sample is blue after addition of the loading buffer, there shouldn't be any trouble, the blue coloration of bromophenol blue corresponds to a pH above 4.6. In case of more acidic pH, the color turns red to yellow... In that cas, you'd better add a few µL of 1M tris solution in order to get the blue color.
Hi Dominique Liger,
Thank you very much, I think your answer must be helpful, and we will do this in our next try. And actually, the sample was truly yellow.
Thank you very much.
Dear Zhichao Lou,
When the pH of your protein mixture is not blue, say yellow meaning the protein mixture is acidic you can overcome this problem by adding few uL of 1M tris that will change the colour to normal blue.