Western blotting-tough question

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Ektor
Ektor's picture
Western blotting-tough question

Hi, i have a question. I am trying to do western using an antibody and ahve tried the recommended concentration. I get a bright yellow band across the gel. The band does not correspond to the molecular weight of the protein i want to detect. It is bright yellow and when i develop the blot it is like it is burned out at that spot. It doesn't have to do with exposure since the band appears after incubation with the primary antibody.....
I have thought it maybe a non specific reaction but the color of the band is strange. Why would it appear so bright?
Does anyone have any ideas?

Thanx

samm
samm's picture
That is very weird indeed. I

That is very weird indeed. I've noticed that when gels are overloaded, (e.g. from tissue lysates), there are some pale yellow-brown bands corresponding to proteins present at highest concentration (by Poinceau staining). However, if a specific antibody is used, only a single band is seen in the xray film. Does your cell express any fluorescent/luminescent protein or any peroxidase class protein?

Ektor
Ektor's picture
I am using brain tissue

I am using brain tissue extract. I think it is what you said. There is non specific binding of some endogenous protein. I will try to use very low protein concentration.

Thanx

ronpbl
ronpbl's picture
If your antibody is non

If your antibody is non-purified polyclonal, it might be due to cross-reacting antibodies in the serum. You might want to try purifying the antibody using an protein A or G resin. Another approach is to prepare an insoluble preparation of your negative control sample. Treating samples with acetone and allowing to air dry will cause them to form as an insoluble precipitate ( make sure it is broken up). This can be mixed with your antiboby stock and incubated for a brief time. Pellet the insoluble sample proteins (and hopefully along with the cross-reacting antibodies). And use the solution for your Western blot. Of course, if you used a monoclonal, or if your antibody cross-reacts with the specific epitope this method will not work. So, if you try this use a little first to see if it works.

nin1318
nin1318's picture
i use mouse and rat brain

i use mouse and rat brain samples from frontal cortex and visual cortex and the only time i have ever seen yellow on a gel is when the dye front gets near the end it changes to yellow...an indication of a change in pH, since the bromophenol blue in the sample buffer is a pH indicator.

perhaps this is what is happening? check to make sure the pH of all your solutions is correct and that you are not running the voltage too high.

Jason King
Jason King's picture
Yellow bands can also occur

Yellow bands can also occur if the extracts loaded on the gel were concentrated via TCA precipitation before hand. I think the TCA protocol includes wash ing the TCA ppt with acetone-HCl then Acetone. Clearly if the acid has not been washed away then the loading buffer will turn yellow and give you a yellow band on the gel. But you would see this even before you loaded the gel.

But I don't understand how you can get a western (chemiluminescent) signal without using a secondary antibody. Are you sure you didn't use an HRP-conjugated primary antibody by mistake?

Philipp
Philipp's picture
Surely, I think if your

Surely, I think if your protein has been loaded much, it could be seen yellowish with a dense degradation form of protein. So it sounds real good to follow the person's opinion down below. Please, load a bit less of protein.