Recently I have been seeing multiple bands in my westerns. I am using the same
method I have been using for years, and it was fine till now. I am using RIPA buffer with protease inhibitors(2X). The protocol for western is even pretty standard. Please suggest if anyone knows whats going on?
Sub Ques: Can Heat cause this? I has started using Bis tris gel and they run at 200V which makes the gel and buffer very Hot.
No this is happening with the antibodies I trust. They were working fine up until now.
The heat maybe is the problem then. If you use ice to cold down the buffer when you transfer your proteins to membrane, maybe it helps.
Hello Mitesh Patel,
I was reading about western blot and I found this information on a website. Maybe it can help you:
- Multiple bands
Cell lines that have been frequently passaged gradually accumulate differences in their protein expression profiles.
Go back to the original non-passaged cell line and run the original cell line samples in parallel.
The protein sample has multiple modified forms in vivo such as acetylation, methylation, myristylation, phosphorylation, glycosylation etc.
Examine the literature and use an agent to dephosphorylatem, etc. the protein to bring it to the correct size.
The target in your protein sample has been digested (more likely if the bands are of lower molecular weight).
Make sure that you incorporate sufficient protease inhibitors in your sample buffer.
Unreported novel proteins or different splice variants that share similar epitopes and could possibly be from the same protein family are being detected. Check the literature for other reports and also perform a BLAST search; use the cell line or tissue reported on the datasheet.
Primary antibody concentration is too high - at high concentration multiple bands are often seen.
Try decreasing the concentration. Run a secondary antibody control (without the primary).
The antibody has not been purified.
Try to use affinity purified antibody. This will often remove non-specific bands.
The bands may be non-specific.
Where possible use blocking peptides to differentiate between specific and non-specific bands. Only specific bands should be blocked (and thus disappear).
The protein target may form multimers.
Try boiling in SDS-PAGE for 10 minutes rather than 5 minutes to disrupt multimers.
You can find more troubleshooting tips here: http://www.abcam.com/protocols/western-blot-troubleshooting-tips.
If your protein sample somehow is digested... sometimes it can happen
Hi Mitesh
I would try the following:
1. Repeat the western if this is the image only from one experiment. You may have skipped the blocking step, or increase the blocking time from 30 mins to 1-2 hrs.
b. Try different blocking buffers - Milk vs BSA blocking buffer.
c. Is your antigen a phosphoprotein and the antibody specific to this phosphoprotein? If yes, then this is expected. Also have you used this Ab previously in western blot runs with recombinant proteins and are now running blots with cell/tissue lysates? Please confirm if "c" is correct and I can explain what is going on and help you trouble-shoot.
Best wishes,
Chandana
Hi,
I think the antibody does not work properly or you have some kind of contamination or unstability in your media. I like Chandana Buddhala suggest to repeat the experiment.
Good Luck, A.
Hi
I think that your protein makes multimer. I had this problem and I examined several sample buffer that one with 8M Urea was good. Also, protein concentration in multimer formation is very important therefore improve it.
Be successful
It looks like primary as well as secondary antibody problem. Try different lot from the same company.
Thanks Neeru, That was I thought. I can overrule primary ab problem as this is happening with more than 3 of my primary abs. I am going to try new secondary abs. Thank you for the suggestion.
I would like to try the Bis tris gel with a cooling system. also I would manage to avoid heating the protein sample before the loading sample, as in a higher tempreture some proteins tend to aggregate thereby forming multiple bands, especially in the case of membane or glycoproteins.
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