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Bands visible in membrane but not in the film


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#1 planktonica

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Posted 25 November 2009 - 02:57 PM

Hi,

I am not very familiar with WB technique (or protein techniques in general). I did a Western and developed yesterday. Today, I was checking something else and realized that there are some visible bands in the membrane that are not present in the film! The bands look exactly the same as the ones on the film, but waay larger (but same pattern and such). I am not expecting large proteins from this western, so I am not clear why this bands are visible on the membrane and not on the film (not that I want them in the film at all!!).

Is that normal?

#2 bob1

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Posted 25 November 2009 - 03:56 PM

A bit of method would be helpful... I am guessing you Ponceau stained your membrane before antibody detection? If so, the bands on the membrane are concentrations of abundant proteins such as cytoskeletal proteins, and may be made up of more than one protein, just similar size/mobility in the gel.

#3 Prep!

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Posted 25 November 2009 - 07:38 PM

hi plank wat exactly do u mean wen u say film??!! if its sumthing to do wioth teh gel.. then tere s incomplete transfer and if the band position is same and only the thickness is teh issue it might just be over loading... as bob says details will be more useful!!!
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#4 planktonica

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Posted 26 November 2009 - 11:55 AM

Hi, thanks for the reply.
I didn't stained the membrane. Just developed after electroblotting, blocking, primary ab, secondary ab... washes... all the standard WB protocol. The film I am talking about is the film used to develop the blotting. I have bands visible on the membrane (never got that before) and they are not in the developed film.

#5 bob1

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Posted 26 November 2009 - 04:01 PM

You didn't pick up a colorimetric kit instead of a luminescent kit did you?

#6 planktonica

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Posted 30 November 2009 - 01:13 PM

We just have luminescent kit :P

My supervisor thinks it is a weird occurrence too...

#7 aimikins

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Posted 30 November 2009 - 02:10 PM

can you post a photo of each, side-by-side?
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#8 medchemgirl

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Posted 01 December 2009 - 02:15 PM

How can you see bands on the membrane if it wasn't stained?? The only bands you should be able to see are the loading marker bands if they are prestained.

#9 madrius1

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Posted 02 December 2009 - 01:11 PM

You can easily see the transferred bands, when you angle the membrane just right under normal light. Also, a dried membrane will make transferred proteins more apparent (almost as clear as a ponceau staining, although not red).

As for the differences, I guess you see total proteins on your membrane, whereas you only detect proteins that interacted with your antibodies, on the film.

#10 medchemgirl

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Posted 02 December 2009 - 02:16 PM

I guess you're right.

You can easily see the transferred bands, when you angle the membrane just right under normal light. Also, a dried membrane will make transferred proteins more apparent (almost as clear as a ponceau staining, although not red).

As for the differences, I guess you see total proteins on your membrane, whereas you only detect proteins that interacted with your antibodies, on the film.



#11 Mary Smith

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Posted 04 May 2010 - 11:59 PM

This is also my concern. Will it be possible to make it visible in the film?




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