How many antibody should be added for Immunoprecipitation - (Feb/02/2006 )

Dear all,

How many antibody should be added for precipitating protein of interest? Waht factors directly affect the amount of antibody used? which factor is most important? Pls help.

-Patrickn-

Hi-

A common starting dilution for IP's is 1:100 into the lysis buffer you are using for IP. That being said it is a smart idea to titrate down the amount of Ab you use per X number of cells. You can run a quick experiment IPing the same number of cells with less and less antibodies and see at what dilution your yield starts to decrease. Obviously, the dilutions may be different depeding on the expression of your protein of interest, thus I usually redo this experiment for each cell type. In my lab, we are cheap (poor!) so I do this every time I switch cell types, try a new induction etc., but if you are just posting on the forum to get a starting point then I would just try 1:100

Good Luck,

Mountainman

-Mountainman-

You need at least 2 ab for an IP followed by Western - one to precipitate with, which you can re-use to probe (athough this is not very elegant) and a secondary. As I have found out if your secondary concentration is too high - you will have a lot of background!

-freyja-

QUOTE (Mountainman @ Feb 3 2006, 12:06 AM)

so I do this every time I switch cell types, try a new induction etc., but if you are just posting on the forum to get a starting point then I would just try 1:100

Good Luck,

Mountainman

Thx Mountainman

for your advice. As your suggested, I should try to use 5 ul of antibody for IP if 500 ul cel lysate was used. However, the concentration of antibodies from different companies is different, so, which amount of antibody (in ng or ug) should I used. Currently, I am using Santa Cruz antibody, which is 200 ng/ul. Do you mean I should use 1 ug antibody (5 ul) for 500 ul cell lysate? On the other hand, How many lysis buffer should be used for a 60-mm dish (confluent) usually? Thx again.

PAt

QUOTE (freyja @ Feb 4 2006, 01:00 AM)

Thx freyja's advice too.

-Patrickn-

Hi,

I've been using Santa Cruz polyclonal antibodies for IP (anti-CBP and anti-Pol II). In general, 5µg of antibody (25µl) in 1000µl of lysate sample works really well. Furthermore, I suggests using a monoclonal antibody against the immunoprecipitated protein to verify the specificity of IP.

Good luck

-Wiz-

Hi-

I don't think it really matters very much what dilution you use for IP's. You will still pull SOME of the protein down. If you want to make sure you're getting all of it without wasting antibodies then you will have to determine that empirically as I mentioned in the other post. It depends on expression level, cell type, lysate protein concentration etc...

I also agree with the others advice too. If available, it's best to use a different detection antibody from your IP antibody to make sure of specificity. I don't know if your IP antibody necessarily needs to be a monoclonal though. I would just see if it lights up a lot of bands when used for western. If so, then it's probably pulling down a lot of other stuff too.

Good Luck

-Mountainman-

QUOTE (Wiz @ Feb 7 2006, 01:14 AM)

Thx Wiz. I will try on your suggested recipe. In fact, I am using the santa cruz anti-p300 (C-20) for IP the endogenous p300 from cells. Could you share with me more experience of pulling down such large protein (i.e. ~300 kDa). Would you mind sharing the protocol with me too? Which antibody for CBP you have used? Thank you very much for your generous help.

In addition, did you measure the cell lysate concentration before IP? Is the cell lysate concentration affect the IP much? I am using 0.75 ml lysis buffer for 60 mm dish. Is it too diluted? or too concentrated? thx

Thx mountainman too. I also want to ask about the effect of cell lysate concentraion. Will the concentraction of cell lysate affect the IP much? thx

-Patrickn-

Here are some references that cite the use of the p300 antibody (C-20) in immunoprecipitation.

You can also search our website at www.scbt.com under "Support": "product citations" to get this same list of citations.

Hopefully you will find this useful for your research!

Yang, C., et al. 1998. A Role for CREB Binding Protein and p300 Transcriptional Coactivators in Ets-1 Transactivation Functions . Mol. Cell. Biol 18: 2218-2229. View PubMed Entry.
Felzien, L.K., et al. 1998. HIV transcriptional activation by the accessory protein, VPR, is mediated by the p300 co-activator. Proc. Natl. Acad. Sci. USA 95: 5281-5286. View PubMed Entry.
Topper, J.N. , et al. 1998. CREB binding protein is a required coactivator for Smad-dependent, transforming growth factor b transcriptional responses in endothelial cells. Proc. Natl. Acad. Sci. USA 95: 9506-9511. View PubMed Entry.
Ogryzko, V.V., et al. 1998. Histone-like TAFs within the PCAF Histone Acetylase Complex. Cell 94: 35-44. View PubMed Entry.
Janknecht, R., et al. 1998. TGF-b-stimulated cooperation of Smad proteins with the coactivators CBP/p300. Genes and Dev. 12: 2114-2119. View PubMed Entry.
Kasper, L.H., et al. 1999. CREB Binding Protein INteracts with Nucleoporin-Specific FG Repeats That Activate Transcription and Mediate NUP98-HOXA9 Oncogenicity. Mol. Cell. Biol 19: 764-776. View PubMed Entry.
Pao, G.M., et al. 2000. CBP/p300 interact with and function as transcriptional coactivators of BRCA1.. Proc. Natl. Acad. Sci. USA 97: 1020-1025. View PubMed Entry.
Marzio, G., et al. 2000. E2F Family Members are Differentially regulated by Reversible Acetylation. J. Biol. Chem 275: 10887-10892. View PubMed Entry.
Ghosh, A.K., et al. 2001. Antagonistic Regulation of Type I Collagen Gene Expression by Interferon-gamma and Transforming Growth Factor Beta . J. Biol. Chem. 275: 11041-11048. View PubMed Entry.
Hasan, S., et al. 2001. Transcription coactivator p300 binds PCNA and may have a role in DNA repair synthesis. . Nature 410: 387-91. View PubMed Entry.
Vandel, L., et al. 2001. Physical association between the histone acetyl transferase CBP and a histone methyl transferase. . EMBO Reports 2: 21-26. View PubMed Entry.
Kim, R., et al. 2001. SNIP1 inhibits NF-kappa B signaling by competing for its binding to the C/H1 domain of CBP/p300 transcriptional co-activators. . J. Biol. Chem. 276: 46297-304. View PubMed Entry.
Schuringa, J-J., et al. 2001. Ser727-dependent transcriptional activation by association of p300 with STAT3 upon IL-6 stimulation. . FEBS Lett. 495: 71-76. View PubMed Entry.
Bradney, C., et al. 2003. Regulation of E2A activities by histone acetyltransferases in B lymphocyte development . J. Biol. Chem. 278: 2370-2376. View PubMed Entry.
Rossow, K.L. , et al. 2003. Synergism between p68 RNA helicase and the transcriptional coactivators CBP and p300. Oncogene 22: 151-156. View PubMed Entry.
Hasan, S., et al. 2002. Acetylation regulates the DNA end-trimming activity of DNA polymerase beta. Molecular Cell 10: 1213-1222. View PubMed Entry.
Mujitaba, S., et al. 2004. Structural Mechanism of the Bromodomain of the Coactivator CBP in p53 Transcriptional Activation. Molecular Cell 13: 251-263. View PubMed Entry.
Narayanan K, et al. 2004. Transcriptional regulation of dentin matrix protein 1 by JunB and p300 during osteoblast differentiation. . J Biol Chem 279: 44294-44302. View PubMed Entry.
Ghosh, A.K., et al. 2004. The tumor suppressor p53 abrogates Smad-dependent collagen gene induction in mesenchymal cells. J. Biol. Chem 279: 47455-47463. View PubMed Entry.
Wu L, et al. 2005. Transforming activity of MECT1-MAML2 fusion oncoprotein is mediated by constitutive CREB activation.. EMBO J 24: 2391-2402. View PubMed Entry.
Treand C, et al. 2006. Requirement for SWI/SNF chromatin-remodeling complex in Tat-mediated activation of the HIV-1 promoter.. EMBO J 25: 1690-1699. View PubMed Entry.

-Immuno06-