Hi, my western blot bands are appearing at the correct molecular weight but are very thin and faint, making it difficult to distinguish difference in expression for shrna samples.
Conditions used:
-50 ug of protein was loaded.
-wet transfer performed
-overnight primary antibody incubation was carried out.
-antibody concentration has been titrated, anything higher gives a black background.
-solvent: blocking & primary antibody: both 5% skimmed milk and BSA in TBST have been tried; secondary antibody-BSA in TBST (concentration has been optimised)
-washing is done in TBST (3*15mins)
-ECL substrate quantity has been titrated.
-This is the most signal achieved after about 20 mins of exposure.
please help!
Sabna Zihara there are often several potential reasons as to why this might be:
I have also provided a link to Abcam's troubleshooting tips for Western blotting - I have always found these very useful!
Hello Sabna
Keeping in mind the conditions you have mentioned I would suggest the following:
1. Protein loading concentration must be 20ug-30ug.
2. Increase the transfer time.
3. Check the power conditions during transfer.
4. Prepare fresh transfer buffer.
5. Reduce the amount of methanol used in the transfer buffer.
6.Use SDS at optimal concentration as it increases transfer efficiency.
All the best.
Hi Sabna, I would do washing 4 times with 5 min each. And I think you should also consider protein amount loaded, 50 ug looks quite high. Thanks
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