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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Western blot with rabbit polyclonal antibodies
Posted by: palche (IP Hidden, New member, 3)
Date: February 22, 2005 09:21PM
Hi. I used purified E. coli protein to generate polyclonal antibodies from rabbit. However, when I am running the western blot, I see no band even though, when stained with commassie, I clearly see the purified protein (should be + control). I use standard protocol for western blocking with 5% dry milk in PBST, primary antibodies (1:2000), secondary antibodies (anti-rabbit, HRP in 1:3000). Will anyone might have any suggestions on what should I change or improve? Thanks a for your time.
Re: Western blot with rabbit polyclonal antibodies
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: March 8, 2005 11:26AM
One way this can occur is if the primary or secondary antibody concentration is too low. It is most likely your primary antibody. Have you tried decreasing the dilution factor? Maybe a 1:1000 or even a 1:500 would work better.
Re: Western blot with rabbit polyclonal antibodies
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: March 17, 2005 07:03PM
Did you raize the antibodies your self or you have aquired it through a service unit? Have they tested for the antibody titer against the purifed protein? If this has been done and you know that the optimal titer for your primary antibody is 1:2000, you need to check for teh follwing:
1- The band you see might not be your pteotin of interest and your might have problems in expression. To eliminate this problem run a purified fraction of your protein if your antibody reacts with your protein then this is in agreement with my former speculation if not it seems your serum has not antibodies against your protein and you need to revise your immunization procedure. 2- If you perform lengthy incuabtion of your Western blot with your primary antibody such as overnight, bacterial coantamination my result in degredation of the antibody resulting in no signal on your Western blot. You need to add sodium azide in 0.05% to inhibit any bacterial growth to your blocking and incuabting buffer/ Good luck!
Re: Western blot with rabbit polyclonal antibodies
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: March 24, 2005 09:45AM
Primary antibody and blocking buffer with 0.05% sodium azide would prevent bacterial contamination. However if you are using chemiluminescene (ECL) to detect the bound antibodies, keep in mind that even very low concentrations of sodium azide will inhibit horseradish peroxidase detection. Therefore you may want to be a little more careful in preforming the wash steps after blocking buffer and primary incubation. With chemiluminescence do not add sodium azide to the wash buffer, as this will result in loss of signal.
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