I didnt add any molecular marker in sample. moreover light bands are seen.
My protein is showing proteolytic activity and fibrinolytic activity too.
But bands are very light .
please tell me what can i do for proper bands.
Before loading samples in your gels, did you quantitate the amount of protein in your sample? Perhaps, the amount of protein you are loading is not enough to help you get the protein profile you are aiming for!
1- You can increase the amount of loaded protein in the gel..
2- Also it's better to reduce the running voltage of the apparatus..
3- don't forget to centrifuge the sample and take the supernatant for loading.
Best wishes
Also you have to use fresh sample buffer with adjusted components for degradation of the protein
I did quantitative assay like Folin lowry and its showing enough amount of protein in my sample. I also concentrated the sample using dialysis method first and then taking the S/N of the dialysate.
How can i increase amount of protein ? The normal concentration of protein:sample loading dye is 20:5 µl .
The voltage applied is 100 V , which is normal for SDS-PAGE.
Thank you for answers.
are you using HRP-conjugated antibodies and ECL or Fluorescent antibodies for detection? If you are using ECL, you can either try using a poly-HRP secondary antibody to amplify the signal or use a really strong ECL such as BioRad's Clarity Max. The Clarity Max is the most sensitive ECL that I have used and it works great. However, it is expensive and will increase the signal strength of all bands. If your antibody is clean with few bands than this works great. If you have a lot of nonspecific bands, this might make the situation worse.
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