Immunoprecipitation - getting rid of antibody heavy/light chains (Jun/19/2005 )
hi all,
i'm trying to to some immunoprecipitations but i need to get rid of the antibody heavy and light chains on my SDS-PAGE gels. Problem is the protein I am interested in is the same size as the antibody light chains, and hence I cant separate it. I have tried Peirce's Seize X kit to cross-link my antibody to Potein G (antibody is a rabbit polyclonal), but i still kept getting antibody eluting off the column. Now i'm trying a do-it-youself cross-linking protocol with DMP. I was just wondering whether anyone had similar experiences/suggestions?
thanks..
-ros-
QUOTE (ros @ Jun 20 2005, 02:31 AM)
hi all,
i'm trying to to some immunoprecipitations but i need to get rid of the antibody heavy and light chains on my SDS-PAGE gels. Problem is the protein I am interested in is the same size as the antibody light chains, and hence I cant separate it. I have tried Peirce's Seize X kit to cross-link my antibody to Potein G (antibody is a rabbit polyclonal), but i still kept getting antibody eluting off the column. Now i'm trying a do-it-youself cross-linking protocol with DMP. I was just wondering whether anyone had similar experiences/suggestions?
thanks..
i'm trying to to some immunoprecipitations but i need to get rid of the antibody heavy and light chains on my SDS-PAGE gels. Problem is the protein I am interested in is the same size as the antibody light chains, and hence I cant separate it. I have tried Peirce's Seize X kit to cross-link my antibody to Potein G (antibody is a rabbit polyclonal), but i still kept getting antibody eluting off the column. Now i'm trying a do-it-youself cross-linking protocol with DMP. I was just wondering whether anyone had similar experiences/suggestions?
thanks..
I believe you will find the same issues using DMP. Crosslinking is not perfect, there is always some degree of variability. But nothing wrong in trying. I could have sworn the Pierce kits use DMP.
-viper-
no they use DSS, but it works the same way (makes amide bonds). thanks for the reply...
-ros-
There is system from Insight Biotechnology called TruBlot that I found helped a little with this problem. The secondary antibody is designed to not recognise the denatured form of the antibody used to IP.
-MicRic-
QUOTE (MicRic @ Aug 13 2005, 02:29 AM)
There is system from Insight Biotechnology called TruBlot that I found helped a little with this problem. The secondary antibody is designed to not recognise the denatured form of the antibody used to IP.
Hi MicRic,
When you say Trublot helped a little, does that mean the IgG heavy and light chains are still visible (but perhaps reduced significantly)? Are you aware of any modifications that can be made (based on your experience) to completely remove these bands so that a protein-of-interest lying in the region can be visualised in its entirety?
The reason I'm asking is that I will probably have to do some Co-IP in the near future and no one in my lab has been able to overcome this problem (i suggested using TruBlot after an internet search, but no one seems to have tried it). Thanks for your time. If anyone else has an answer to my question, pls feel free to share it.
Regards,
ggUss
-ggUss-
QUOTE (ggUss @ Aug 12 2005, 09:00 PM)
Hi MicRic,
When you say Trublot helped a little, does that mean the IgG heavy and light chains are still visible (but perhaps reduced significantly)? Are you aware of any modifications that can be made (based on your experience) to completely remove these bands so that a protein-of-interest lying in the region can be visualised in its entirety?
The reason I'm asking is that I will probably have to do some Co-IP in the near future and no one in my lab has been able to overcome this problem (i suggested using TruBlot after an internet search, but no one seems to have tried it). Thanks for your time. If anyone else has an answer to my question, pls feel free to share it.
Regards,
ggUss
When you say Trublot helped a little, does that mean the IgG heavy and light chains are still visible (but perhaps reduced significantly)? Are you aware of any modifications that can be made (based on your experience) to completely remove these bands so that a protein-of-interest lying in the region can be visualised in its entirety?
The reason I'm asking is that I will probably have to do some Co-IP in the near future and no one in my lab has been able to overcome this problem (i suggested using TruBlot after an internet search, but no one seems to have tried it). Thanks for your time. If anyone else has an answer to my question, pls feel free to share it.
Regards,
ggUss
I can't find the blots I did with TruBlot. I stopped doing IPs recently because of a mix up with the antibodies I had. I should be starting them again in the next two weeks. My memory is that there was a massive improvement over what I had seen with Protein A and Protein G. I will be starting up again with the IPs as soon as our X-OMAT is fixed and I'll report back what I find.
-MicRic-
QUOTE (MicRic @ Aug 17 2005, 05:52 PM)
QUOTE (ggUss @ Aug 12 2005, 09:00 PM)
Hi MicRic,
When you say Trublot helped a little, does that mean the IgG heavy and light chains are still visible (but perhaps reduced significantly)? Are you aware of any modifications that can be made (based on your experience) to completely remove these bands so that a protein-of-interest lying in the region can be visualised in its entirety?
The reason I'm asking is that I will probably have to do some Co-IP in the near future and no one in my lab has been able to overcome this problem (i suggested using TruBlot after an internet search, but no one seems to have tried it). Thanks for your time. If anyone else has an answer to my question, pls feel free to share it.
Regards,
ggUss
When you say Trublot helped a little, does that mean the IgG heavy and light chains are still visible (but perhaps reduced significantly)? Are you aware of any modifications that can be made (based on your experience) to completely remove these bands so that a protein-of-interest lying in the region can be visualised in its entirety?
The reason I'm asking is that I will probably have to do some Co-IP in the near future and no one in my lab has been able to overcome this problem (i suggested using TruBlot after an internet search, but no one seems to have tried it). Thanks for your time. If anyone else has an answer to my question, pls feel free to share it.
Regards,
ggUss
yeah, that will be great.
I can't find the blots I did with TruBlot. I stopped doing IPs recently because of a mix up with the antibodies I had. I should be starting them again in the next two weeks. My memory is that there was a massive improvement over what I had seen with Protein A and Protein G. I will be starting up again with the IPs as soon as our X-OMAT is fixed and I'll report back what I find.
-ggUss-