Hi Bardia Varastehmoradi,

First, it is important to make sure that you are looking to the right band. What is the molecular weight of the glucocorticoid receptor?

The multiple bands in the region you marked can be different isoforms of this receptor. Is the antibody monoclonal or polyclonal? In the case of polyclonal, multiple bands are common. Are you interested in one specific isoform only? If yes, you will have to identify which band corresponds to the isoform of interest and quantify it alone.

Sometimes, different isoforms are equally important to measure the activity of the protein, and you can quantify all the bands. This is the case of ERK 1/2, for instance. They were both important for ERK pathway and my antibody detected two bands. I quantified the area surrounding both bands and excluded the background signal.

I hope this helps,

Priscila

Hi Bardia Varastehmoradi,

First, it is important to make sure that you are looking to the right band. What is the molecular weight of the glucocorticoid receptor?

The multiple bands in the region you marked can be different isoforms of this receptor. Is the antibody monoclonal or polyclonal? In the case of polyclonal, multiple bands are common. Are you interested in one specific isoform only? If yes, you will have to identify which band corresponds to the isoform of interest and quantify it alone.

Sometimes, different isoforms are equally important to measure the activity of the protein, and you can quantify all the bands. This is the case of ERK 1/2, for instance. They were both important for ERK pathway and my antibody detected two bands. I quantified the area surrounding both bands and excluded the background signal.

I hope this helps,

Priscila

The proteins in the bands are different enough that they separated in your blot. You may not want to combine different bands from the same blot (or lane) that are well separated. However, you may have to combine similar bands from different blots or lanes that look identical, in order, to get enough material for further study. Please consider that the Western Blot has helped to 'purify' the proteins in the bands, so don't give up this desirable result. I am not sure how your separation is based, but if separation is mostly by molecular weight as in PAGE, you may want to run a lane of molecular weight standards, simultaneously, if you did not do so.

Hi,

Adding to what is yet answered, was your antibdy already cited? Could you refer to these reference? Do you have a positive control (purified phospho glucocorticoid receptor for instance) and a negative one (purified non-phospho glucocorticoid receptor or extract without phospho inhibitors)? Did you another antibody?

If you want to know what is precisely in these band, you shall do MS/MS.

Selection via correct MW should be the easiest way to select the correct band in WB analyses. Moreover, you should use the correct band (not all in combination, as also mentiond before).

Hi,

As others have suggested, I would:

1. If possible, check how specific is your ab (negative/positive control). As this is usually difficult, I would:

2. Check the MW

3. Find other articles that have used the same ab from the same company

4. Select the specific band for your ab and quantify. You can´t select the whole area, as you don´t know what you have there.

Good luck!

Thank you all for your answers. they are really helpful. My samples were not collected from human. They are homogenized samples of different brain regions originated from rats( Sprague Dawley).

have you intented to do a gradient gel to separate the bands or Isoforms? Check the specificity of your Ab. Or chance the concentration of the gel. Maybe you can isolate protein membranes and citosolic.