Hi Muluken,

If I understand you right, you would like to determine the M.W of the two bands so as to compare them and know which one is the right band and which might be unspecific/phosphorylated/ubiquitinylated and so on. as you mentioned that the bands are very close, i would first try to run on a higher percentage gel (so as to increase the separation between them) and also load perhaps two markers (a low range and a high range). then I would also load a control (say GST:25kd or a known protein around the same size as your size, antibody anti-gst) in one lane. and then measure the relative migration of the proteins on the gel. loading two markers will help you pinpoint the exact weight your protein is running on the gel.

Best,

Gurjot

Dear Muluken Mamo,

Though I am not experienced in working with BDNF, in general for western blotting as a technique per se  I can suggest you the following:

Molecular weight calculations can be done by relative migration method of your markers(Prestained ladder) and then by regression analysis you can easily estimate molecular size of your band of interest.

What about the specificty of your primary antibody?If it is very specific then be assured that the upper band is nothing but the phosphorylated or ubiqitylated form of your protein of interest.

Dear Muluken Mamo,

Western Blot gives you approximations of molecular weight, but is nothing good in determination of exact MW. This is because beside of MW, also netto loading is affecting the running characteristics during SDS page, ie, a single phosphorylation  can induce a profound shift in your band.

Beside of the aforementioned specifity issue of the antibody, you may want to check weather you see a phosphorylation by comparing lambda-phosphatase treated versus non-treated lysates and see if the upper band disappears.

If you want to have an exact molecular weight, you have to go for some mass spectrometry (MALDI-TOF) mesurements.

Use an LC -MS..and MS/MS will be perfect to identify not only the molecular mass..you will identify the amino acids sequence and possible post-translational modification which usually change the native protein mass.  Good luck ! Alina

Hi Muluken,

If I understand you right, you would like to determine the M.W of the two bands so as to compare them and know which one is the right band and which might be unspecific/phosphorylated/ubiquitinylated and so on. as you mentioned that the bands are very close, i would first try to run on a higher percentage gel (so as to increase the separation between them) and also load perhaps two markers (a low range and a high range). then I would also load a control (say GST:25kd or a known protein around the same size as your size, antibody anti-gst) in one lane. and then measure the relative migration of the proteins on the gel. loading two markers will help you pinpoint the exact weight your protein is running on the gel.

Best,

Gurjot

The EXACT weight of proteins may be established ONLY by MS ...not by SDS / or WB even using standard protein markers....

Dear Muluken Mamo 

An easy way to approximate the molecular weight of a particular protein is by a logarithmic regression analysis, you can plot log(mw) vs relative mobility, with this information you can intrapolate with the respective mobility of the bands and obtain the approximate molecular weight. 

I hope you can find it useful. 

I'm sorry that the topic of my question is misleading  but I am not highly interested in measuring the exact molecular weight of the bands. I just wanted to know which of the two bands represent BDNF. Thank you everyone for your answers.

 Dear Gurjot Kaur, that will help a lot. I am going to try it soon.

Hi,

1. Did you get two bands in the input as well (like whole lysate) ??

2. If you have antipeptide against your antibody then you can try with that and see which band is disappearing. If both the band are disappearing then it could be they protein is dimerized or degraded. 

good luck and keep us updated

Mamo,

The two bands could be your protein of interest that may have undergone post translational modification such as phosphorylation or palmitoylation provided your antibodies are specific. On the other hand, the lower band can also represent proteolytic breakdown product of your protein. Hope you used appropriate protease inhibitors during lysate preparation and you avoided repeated freeze thaw of samples.

Hi there,

What is the antibody used for the detection? If anti-BDNF then both bands contain motifs recognized by the antibody. Then analyze the bands by MS peptide mass fingerprint to get which corresponds to the intact BDNF

here is an example

https://info.gbiosciences.com/blog/bid/196974/determining-protein-molecular-weight-with-sds-page-an-overview-of-the-process