I am using DAB staining to detect signal for protein expression, but now a days I am getting faint bands? Even increased the protein concentration, fresh antibody dilution, reduced blocking steps etc., can anyone suggest be how I can overcome this?
Lakshmikanthan, can't say I have ever heard of DAB being used for WB. I have only ever used DAB for IHC. I would highly recommend many of the great chemilluminescent subtrate reagents available commercially.
Improving sensitivity of WB requires consideration of many different factors (too numerous to state here). Not knowing the details of your procedure I can only recommend the many resources (online and at your library) available to you that I believe will adequately answer your question. To get you started I have included a link of a PDF available from Abcam. I hope it is useful to you.
In general, I can only recommend that you replace all of your reagents (solutions and antibodies) and start with fresh material. Insure that your protein lysates are sufficiently protected against proteases by adding a molar excess of protease inhibitor to your lysis buffer.
Best.
http://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf
DAB is an old technique. Try checking pH of Tris whether it is 7.6. This pH is very critical for DAB in WB. Use 3, 3' DAB. There are additional DABs which will not work for WB. Reduce wash times to 3 mins each. Finally, there are certain proteins always expressed less. Confirm the staining procedure using actin.
Check the activity of the secondary. And you can enhance a bit the DAB staining adding Co2+ (or Ni2+) (1%) to the staining solution. Do not use phosphate buffers.
Try acetate immidazole buffer just for last step to prepare the substrate..
Check your hydrogen-peroxide, is really good enough to react. Try with new H2O2. !!
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