Hi Rachel,

1) That will depend on protein X expression. Without tests its hard to tell.

2) By native, do you mean without SDS ? If you can't detect the protein it might indicate that the epitope recognized by your antibody is not accessible by your antibody when protein is native (hydrophobic regions deep inside the floded protein for example), while when denatured epitope is exposed. You might test that by adding SDS in your three native buffers and see if suddently the protein band appears.

3) Yes, if you can't detect protein alone, you won't see any interaction :)

Hi Rachel,

1) That will depend on protein X expression. Without tests its hard to tell.

2) By native, do you mean without SDS ? If you can't detect the protein it might indicate that the epitope recognized by your antibody is not accessible by your antibody when protein is native (hydrophobic regions deep inside the floded protein for example), while when denatured epitope is exposed. You might test that by adding SDS in your three native buffers and see if suddently the protein band appears.

3) Yes, if you can't detect protein alone, you won't see any interaction :)

Thanks! Alexis. One of buffer we tried contains 0.1% SDS. We will try adding more SDS and see how it goes.

You might try to extract your protein(X) in RIPA buffer and add antibody as usual. Incubate O/N at 4 C. Add Protein- A-Sepharose beads to it. Incubate it for 2-3hrs

( better O/N at 4 C). Wash 5-6 times with cold PBS containing 0.1%TritonX100 / NP40. Elute the immune complex with 25mm Tris-HCl pH2.5 or you can use 3M NaSCN( a chaotropic agent) to release the immune complex from the beads.For NaSCN, you need to dialyse the eluent to get rid of NaSCN. Spin, concentrate and run the gel. Good luck.!

Rachel:

First, congratulations on working at UC Berkeley. I grew up in that city and still miss it and the UC campus.

(1) I did my dissertation work on a maize cell wall protein. I may not understand your first question. When you ask how much starting protein (extracted from maize anthers) should you try first, I ask how much you used to obtain your pAbs. All things equal, if you can get 10 mg almost as easily as 1 mg, why not use that? You will have more material on which to work.

(2) One of the problems with a Western Blot from SDS-PAGE is that the epitope is probably denatured. Increased [SDS] means increased denaturation. This is important: Did you induce pAb production with native protein X or with your protein X solubilized with SDS? If the pAbs were developed in response to native protein, try to run an IEF gel and try the technique in this paper: Johnson, T. K, Yuen, K C. L., Denell, R. E., and Consigli, R. A. Efficient transfer of proteins from acetic acid-urea and isoelectric focusing gels to nitrocellulose membrane filters with retention of protein antigenicity. Anal. Biochem. 133(1): 126–131 (1983). I used their technique successfully. I assume from your test that your protein X is not a membrane protein. If it is, read the thread on "Membrane Protein Western Blot" for more information.

The protein may be soluble but denatured in your buffers. What buffers are you using? Protein work can be very sensitive to interferents. Are you rinsing your glassware with at least 3–6 M HCl after washing? Are you filtering your buffers before use? I did not filter _sterilize my buffer stocks, but I did filter "clean" them with a filter assembly. It was cheaper than FS'ing and I never saw growth in my buffers. BTW, you should have seen the filters... Also, if your buffers have a pH of > or = 7.5, do not use nitrocellulose filters; they will break down.

(3) Yes, if you can not detect protein X in your starting sample, you have a problem with the sample, with your pAbs, or both. You should be able to run a gel and Western that shows increasing concentration of your protein and decreasing concentration of everything else with each purification step. Run two lanes of each step, then stain one set of lanes with Coomassie Brilliant Blue or whatever you prefer and another with you pAbs and a conjugated secondary Ab.

(4) How are you purifying your samples, or have you gotten there yet?

Wales

To answer ur question need more information about ur protein preparation.

you just check with the following steps.

Increase the amount of protein loaded per lane. Verify solubilization and extraction protocol used on biological sample. Titer total protein loaded until signal appears.

Confirm the lysis buffer used was strong enough to disrupt the cell’s membrane, nucleus , etc., where the target is localized. Use appropriate protease inhibitors to prevent degradation.

Use 0.2 µm membranes instead of 0.45 µm to prevent transfer through the membrane.

Add SDS to the transfer buffer and lower the concentration of methanol.

Thanks! Amit. We have tried RIPA buffer, but I am worried that our protein is insoluble in RIPA buffer.?? Because after protein extraction using RIPA buffer, we run a SDS-PAGE gel and did a western blot with X antibody, we cannot detect our protein X as usual. (we regularly extract protein by acetone precipitation method, and have no problem to detect our protein X on western blot). I am thinking we might have a small window to have soluble protein X and complete protein complex...

Thanks! Wales.

I love Berkeley city and UCB.

1) for our western in general, we run 30 ug protein extracted from maize anthers using acetone precipitation method on SDS-PAGE, we are able to detect a sharp/clear band by 1:5000 dilution of our antibody X. But when we run 40 ug protein extracted from anthers using either RIPA buffer or other IP buffers, we fails to detect any bands. ON the first attempt, we used 600 ug as starting material for IP, but did not get any interesting results. I think you are right, more is always better for IP.

Thanks for the ref and information. Our protein is a nuclear protein. We will check these points you mentioned. Thanks~~

Thanks! Vakamullu. Our protein is a nuclear protein. I guess the lysis buffer is too mild to disrupt nuclei or our protein is insoluble in the lysis buffer. We will try more harsh buffers first. Thanks!

Hi rachel another offer from me is that if you are trying to pull-down a nuclei protein firstly it is possible to use related method that you can purify nuclei directly thus you can eliminate irrelevant protein coming from cytoplasm thereby you can just get your interested protein from nuclei even by mild buffer, i was in same point with you and with using this method i managed it! Good luck in advance..

Rachel, I should have asked this earlier: What protease cocktail are you using?

Funny: Between Cenk and me, you have a couple of Turks trying to help.