Hello Dear Friends,
I have experienced a problem in the pAKT protein expression, it shows white color band. I have used recently purchased santa cruz antibody, primary antibody (1:1000 dilution and secondary 1:5000 dilution). so, what would be the reason, please give your expertise and comments on this.
Please find the attachment file bellow.
Thanks in advance for your valuable suggestions and comments.
Cannot see well the blot (low quality of the image) White bands occur when the reagent is exhausted due to excess of signal (or of amount of assayed the protein) but at so high dilutions of Abs it should not be occurring.
Are you showing pAKT or total Akt? Is total AKT white also?
Dear Dr Franco,
Thanks for your valuable comments. The total AKT is not white in color, the pAKT alone showed white color band pattern.
Which is more advisiable should increase primary antibody dilution or secondary antibody dilution?
Hi,
I agree with Rafael, the excess of signal or long exposure is the main reason.
Have you used the ECL detection? Probably reduce the volume and try another batch of ECL.
Hope that helps.
Hello Dr Stephane
Yes, I have used ECL detection .. I will follow your instruction. Thank you for suggestion.
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