"Westerning" your self out... - (Aug/09/2005 )

Hello to all of you.

Here is my problem. I am trying to perform a western blot in order to detect my protein in HEK 293 lysates. I have a positive control which works just fine. The positive control is lysates from mouse tissue. (as I mentioned my samples are human). I use a Santa Cruz ab. The problem is that I don't get the desired band although it seems that there is nothing wrong with my western. My ponseau is very good, my tubulin is very good too. I don't know what to do. I thought that it would be good idea to change my blocking buffer. I used blocking 5% dry milk for 1-2 hrs, and 2% dry milk for diluting my primary ab. I changed it to 1% BSA for diluting my ab. The result was that I got white bands (instead of black) that they seem to be what I want...but I would not bet my head on it because the signal is extremely low.

Any ideas guys?

Thanks a lot!
Maria Eua

HI!
What detection system are you using? and what kind of protein are you trying to detect?

Goodmorning molbionovice,

I am using ECL from Amersham. I am trying to detect a cysteine aspartase.

goodmorning to you dk_actrec,

The ab seems to work ok because my positive control is ok. The white bands although they are in the right molecular weight seem to be a background because they also appeared when I probed my membrane with another ab. My desired protein should at 55 to 43 kD.
Yes the positive control gives the correct ones.
So there seems a problem with antibody or tissue that you are extracting. What do you mean, what could go wrong? They are HEK 293 cell lysates and I suppose that if something was wrong with my samples I would see it (degrated proteins in my Ponseau, or bad tubulin signal, right?)
Yes, my ab recognize human rat and mouse so it should give me my bands.
Any other suggestions?

Thanks guys for your replies.

Hi Maria

I have read that white bands on a dark background may be because of too much HRP in your system. Why don't you try reducing the conc. of your secondary=HRP tagged antibody?

Another way to go about the problem would be to try to eliminate your background step-by-step as explained below;
After you run your gel and transfer to a membrane, cut your lanes out and try each with the foll conditions:
strip 1- no blocking, do primary, secondary and ECL
strip 2- blocking+no primary+add secondary+ECL
strip 3- blocking+primary+no secondary+ECL

This should help you find out where your background is coming from.

All the best!

Hi Maria,

I get the result if I use way too much secondary. So I will have to agree with molbionovice that the white bands on your Western are due to you using too much of your secondary Ab. Try reducing it by at least 2 folds (i.e. instead is 1:2500 try 1:5000).

HTH

Jeff

Dear jeng and molbionovice,

Thanks for your kind help. I think that my major problem is not to eliminate my background but to see my desired bands. I had tried in the past less concentrated secondary ab (and primary) and since I got nothing I thought to make my ab more concentrated. So either I have no bands at all (a completely clear membrane) or I get background. Would it be possible that the mouse human thing (among my positive control and my samples) reguire a different protocol optimization from the scratch? I mean different incubation times, try different blockings (FBS, BSA, dry milk) and different concentrations of them etc?
Nevertheless I will try your idea molbionovice, to see what happens.

P.S. I suppose that loading 30-40 μg of proteins are enough right? I am thinking of increasing the protein amount but I don't think it would make any difference. Do you?

Thanks a lot!
Maria

Hello !

"I suppose that loading 30-40 μg of proteins are enough right? I am thinking of increasing the protein amount but I don't think it would make any difference. Do you?"

That can be the reason!!!
I read in some published studies it is possible to load approximatelly 5 up to 200 micrograms !
And I don´t know if more or less. It should respect loading capacity, so you need to adjust (to concentrate) your sample.
(For example at IRS-2, Akt proteins…)
Check it in method in some studies specially for required protein.

It depends on tissue you have and others as with combinations of used …

tissue - primary, secondary antibody (kind of supplier company) - detection system - imager system…

It can be really different. It really depends on your own lab procedure and you need adjust it for. Literature is only suggestion.

If you need more (micrograms) protein and you can´t more concentrate your sample to load for example 200 micrograms, you can use immunoprecipitation = to extract only your protein from your lysate.