Hello!
After harvesting my cells, I resuspended them in RIPA buffer and stored them at -80°C. After determining the protein, I usually load 10-25 µg onto a 4-15% SDS gel. The protein on the gel is transferred to a membrane using the semi-dry method. The membrane is blocked with 5% BSA for one hour, then I applied various antibodies dissolved in 5% BSA and incubated it for at least 2 hours or overnight. I incubated the secondary antibody for 1 hour. In total I used 8 different primary antibodies. With almost all antibodies, I get numerous other non-specific bands in addition to the expected band (see appendix). The primary and secondary antibodies were diluted 1:1000. The Immubilon Forte Western HRP Substrate from Merck was used for detection by chemiluminescence. Does anyone have any idea what I could change or what might be the reason I'm getting so many unspecific bindings?
Thank you for any tips!
Hello,
You can try to decrease the concentration of primary ab and/or secondary ab.
What is the cat. number of primary and secondary antibodies? I would like to do a search a little. And what is the source of your cells? Which cell line or which species?
From your content, it seems that the several problems could be involved to generate such non-specific banding pattern on the membrane in WB:
1. Check that all primary antibodies are monoclonal or polyclonal
2. Check the source of primary antibodies
3. Check the cross-reactivities of all primary antibodies
4. Block the membrane with 5% BSA or with goat milk overninght
5.Try to dilute the primary antibodies concentration to (1:2000)
6. Expose with primary Ab for 2 hrs only
7. Wash thoroughly membrane with wash buffer to remove the traces of primary antibodies from membrane
8. Check that the secondary Ab is developed against all primary antibodies source
If the problem still persist then expose the membrane with only one primary Ab to avoid any other possibility due to multiple Ab interactions on the membrane.
Hope, these changes shall help you to get desires bands only
Good luck
Lowering primary/secondary antibody incubation time along with an increase in blocking time have been very helpful for me so far. Good luck.
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