Co- IP of 53 KD proteins - Immunoprecipitation (Oct/28/2007 )
Hello,
I would like to perform a Co-Ip for two proteins about 53-55 KD. However, I am concerned the result will be masked by the IgG because it runs at the same molecular weight.
1) How can I avoid this issue?
2) If I am going to use mouse monoclonal antibodies for both, is it better to use A- sepharose beads or can I use A/G sepharose beads.
3. What is the difference between agarose beads and sepharose beads.
4. Which is the best company to buy these beads.
Thanks
-austen-
QUOTE (austen @ Oct 28 2007, 03:19 PM)
Hello,
I would like to perform a Co-Ip for two proteins about 53-55 KD. However, I am concerned the result will be masked by the IgG because it runs at the same molecular weight.
1) How can I avoid this issue?
2) If I am going to use mouse monoclonal antibodies for both, is it better to use A- sepharose beads or can I use A/G sepharose beads.
3. What is the difference between agarose beads and sepharose beads.
4. Which is the best company to buy these beads.
Thanks
I would like to perform a Co-Ip for two proteins about 53-55 KD. However, I am concerned the result will be masked by the IgG because it runs at the same molecular weight.
1) How can I avoid this issue?
2) If I am going to use mouse monoclonal antibodies for both, is it better to use A- sepharose beads or can I use A/G sepharose beads.
3. What is the difference between agarose beads and sepharose beads.
4. Which is the best company to buy these beads.
Thanks
your protein of interest is exactly at the levels of Ab heavy chains to avoid this
- you should use antibodies raised from 2 different species, if u precipitate with mouse monoclonal then detect your Co-ip by rabbit or goat Ab.
- ebioscience has a secondary antibody which detects only the intact primary antibodies and not the cleaved ones(more info chk their website) i tried it and works ok..
- i use magnetic protein G beads from miltenyi biotec so cannot advice for your 3rd and 4th questions.
hope this helps
rajgene
-rajgene-
QUOTE (austen @ Oct 28 2007, 07:19 PM)
Hello,
I would like to perform a Co-Ip for two proteins about 53-55 KD. However, I am concerned the result will be masked by the IgG because it runs at the same molecular weight.
1) How can I avoid this issue?
2) If I am going to use mouse monoclonal antibodies for both, is it better to use A- sepharose beads or can I use A/G sepharose beads.
3. What is the difference between agarose beads and sepharose beads.
4. Which is the best company to buy these beads.
Thanks
I would like to perform a Co-Ip for two proteins about 53-55 KD. However, I am concerned the result will be masked by the IgG because it runs at the same molecular weight.
1) How can I avoid this issue?
2) If I am going to use mouse monoclonal antibodies for both, is it better to use A- sepharose beads or can I use A/G sepharose beads.
3. What is the difference between agarose beads and sepharose beads.
4. Which is the best company to buy these beads.
Thanks
In addition to choosing different species for the antibody or using the trueblot, you can also covalently couple your antibody to the beads using a cross linker like dimethyl pimilimidate.
Sepharose is I think internally cross linked agarose.
If you are unsure of which protein A or G sepharose to use look at a chart online using a google search. Certain antibody isotypes bind poorly to one but great to the other. For instance IgG1 binds great to protein G but not so great to protein A. This also varies accross species....
-Mountainman-