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Incomplete Blotting and staining of NC membrane

Western Blot ELISA Blotting Incomplete Problems

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7 replies to this topic

#1 TZ89

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Posted 25 January 2014 - 03:08 AM

Hello everybody,

 

I'm trying to run an SDS-PAGE with a Western Blot. The SDS-PAGE is working fine so far, but I'm not sure if the Blotting really works. I'm using Nitrocellulosis and run the gel at 100 V for 1 h.

To prevent overheating of my Transfer buffer, I'm using cooling pads in and around the chamber. After 1 h I see the bromophenol blue of my prestained marker on the NC Membrane. When I now stain the Membrane with Ponceau S I cannot see any bands on the NC. (That could possibly be caused by too low concentration? So low that it is under the detection limit of the Ponceau method?).

 

In the following I'm trying to make the bands of interest visible. For that I'm using materials of an ELISA-kit. In detail, I have an antibody on the NC membrane, I'm giving specific antigen (from the ELISA kit) on the NC, then I'm adding specific biotinyled detection antibody and strep-HRP (also from the ELISA kit). When I'm now adding TMB (HRP-Substrat) the solution turns blue step by step but I cannot see any changes of the NC membrane itself. The positive control I'm using is primary antibody (5myg/ml), Antigen (1 ng/ml), secondary AB (1 myg/ml).  

 

Is it possible that maybe only the blotting of bromophenol blue of the marker works but because of any reason no Protein is blotted? Is the voltage or time of bllotting too high/low?

 

Thank you very much

 

TZ89   



#2 jerryshelly1

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Posted 25 January 2014 - 10:39 AM

When you are finished with your transfer, stain your gel with commassie to see if any residual protein remains on the gel. Either your proteins are not transferring or your proteins are blowing through your membrane.



#3 mdfenko

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Posted 25 January 2014 - 02:53 PM

if the protein is large then it may not transfer well without modification of the transfer method.

 

what is the pore size of your membrane?

 

formulation of your transfer buffer?

 

size of your protein of interest?

 

did you see the pre stained protein standards on the membrane (besides the bromphenol blue) or were they still in the gel?

 

as jerryshelly1 said, you should stain the gel to determine if the protein transferred. you should also place a second membrane behind the first to determine if you blew the protein through the membrane also look at the paper backing the membrane to see if the pre stained standards blew through.


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#4 TZ89

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Posted 27 January 2014 - 01:58 AM

Thank you both for your help. The pore size of my NC Membrane is 0,2mym. My transfer buffer has the following formulation:

V = 1 L; 48 mM TRIS; 39 mM Glycin, 0,037% SDS; 20% v/v Methanol; pH=8,7

The protein I'm looking for has an unknown size. Its only kown, that it interacts with a cytokine. We suppose that its size range between 3 and about 200 kDa. For that I'm running a 12% SDS-gel. When I make a Ponceau staining I cannot see any red stained bands at the blue bromophenol blue bands.

 

I think it will be the best, when I repeat the transfer as I did before but with two membranes, with the same marker and some known proteins. When I stain the gel with commassi and the two membranes with Ponceau, I hope that I will see some bands somewhere.   



#5 Missle

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Posted 27 January 2014 - 05:20 AM

Your pre-stained markers should transfer nicely to your membrane and be a quick sign that your transfer happened.  Their presence on the gel is also a quick sign that it didn't.  What you've outlined above is a good strategy but using prestained markers and listening to what they are telling you will give you the quickest answer smile.png  If everything is fine with your marker then you will need to continue on to evaluating what about your protein is making it not transfer well but troubleshooting the actual process is a good first step.


Edited by Missle, 27 January 2014 - 05:22 AM.


#6 TZ89

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Posted 28 January 2014 - 10:41 AM

I have tried to find protein on the membrane or gel. But sadly there's nothing. But I noticed that there's a red area (from Ponceau S stain) on the side of my membrane (that side that is on the lower end of my western blot cassette). It looks like proteins are gathering at that end. Could that be a sign for too high voltage or time during transfer?



#7 mdfenko

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Posted 29 January 2014 - 04:24 AM

are you sure you have good and complete contact between the gel and membrane? it sounds like the protein leaked between them.


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#8 TZ89

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Posted 25 February 2014 - 08:12 AM

I now have run a successful Protein Transfer on my western blot. It was possible by using Methanol activated PVDF Membrane at a voltage of 50 V for 150 min. Thank you all for your help!







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