Antibody-Biotinylation, detection via SDS-Gel - (Feb/02/2015 )

Hey forum members

 

I have some problems to interpret my SDS Gel of an an Antibody which is conjugated to Biotin-NHS.

 

I tried to show biotinylation of an Antibody ( IgG, anti Digoxygenin) with coupling of streptavidin and SDS PAGE(non reducing, non denaturating conditions).

 

The idea was to get a higher molecular weight when coupling the biotynilated Antibody to streptavidin.

 

 

The Problem is, that my SDS Gel shows multiple bands by adding just Biotin NHS.

I acctually thought that when I ad biotin NHS to the antibody that there will be no detectable changes at the appearing bands.

But there are bands which are below the band of the native antibody, which I couldn`t explain.

 

I thought of Biotin Biotin interactions because of Hydrogen bonds but this doesnt explain why they are lower than the native band of the antibody.

 

When streptavidin was added( 6µM) you can see just a little change in the intensity of one Band (I acctually thought the effect would be much higher)

 

You can also see lots of unbound streptavidin at this sample.

The sample without Biotin NHS but with streptavidin shows no unbound streptavidin which is also confusing.

 

 

(I worked with a 20 fold molar excess of Biotin NHS) the reaction with the antibody was allowed to proceed for 2h on ice.

After biotynilation, I dialysed the antibody against(20mM Hepes pH 7,4) to remove unbound Biotin-NHS.

 

I hope someone can help me

It would be great if you could tell me why there are appearing bands after adding just Biotin NHS.

 

I just need to know if my biotinylation was successfull

 

Thanks a lot.....

 

 

 

 

 

 

a couple of things:

 

what is your gel formulation (including buffers). you refer to it as "sds-page" then say it is non-reducing and non-denaturing. did you completely omit sds from the gel and buffers? if so then just call it page or native page. if not then it is denaturing.

 

with native page charge becomes more important than size (within the limits of the restrictiveness of the gel).

 

if the protein markers were prepared for sds-page then they won't be valid for native page. if they are for native page then their size distribution may not be accurate. you can enhance size separations with a gradient gel.

 

multiple bands may be due to variable incorporation of biotin (biotin-nhs will bind to available primary amines, eg lysines) and their effect on charge.

 

you do show some faint bands above the main band with the addition of streptavidin.

 

also, your sample load is too generous. it's easier to see differences with finer banding. you may even be overshadowing the streptavidin bound antibodies.

 

just somethings to think about.

the sds may also cause dissociation of the complex. you probably should use a native gel to ensure that the complex remains together.

Is it possible, that Biotin-NHS causes the multiple bands because of charge effects although I used SDS?

I don't know, If I understood your experiment correctly, But some thoughts-

 

Can't you use western blotting to probe if biotinylation has happened or not? If you do the same gel, and probe with avidin or streptavidin antibody?

 

I agree with mdfenko, that you should do a native PAGE, anyways you are adding only SDS, instead add CBB G250 and do a BN PAGE.  

petertob on Tue Feb 3 07:29:25 2015 said:

Is it possible, that Biotin-NHS causes the multiple bands because of charge effects although I used SDS?

it may not be a charge effect but, rather, a size effect based on a variable number of biotins bound (although, if the biotins bind at a sds binding site then there may be an effect on charge).

there may also be an effect from variable unfolding by sds treatment.

 

also, if you want to determine size differences without dissociating the complex then bn-page (as neuron suggested) may be the way to go.

Hmm ok I understand,

I just thought when Biotin is added then there need to be a higher mass so the bands should be above the native antibody.

 

I am just confused because the bands which apeared with addition of Biotin-NHS are lower then the native antibody.

 

I am sorry I am just studying and I have not so much experience yet.

biotin doesn't increase the mass by much (for each one). it may be altering the charge of the protein so that it migrates faster than untreated protein.

 

i used to phosphorylate proteins and then run them on urea-page (to enhance charge separation). the phosphorylated proteins ran faster than the non-phosphorylated proteins despite the increase in size and mass.

AH ok that sounds logical  

Thx mdfenco!!!

Does anybody know if its possible to store biotinylated antibodies (biotinylated with Biotin-NHS) when they are not lyophilized?