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western blot


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5 replies to this topic

#1 yyang

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Posted 11 January 2005 - 04:40 PM

Hi,
I am doing a western blot on the claudin-5, one of tight junction proteins. The protocal I am using worked very well at first several stainings and I got the specific bands with right molecular wieght. Then there were no bands found and the films are totally blank without a bite of background when I repeated the blotting. I checked everything that was supposed to give rise to the troubkes. I found everything is ok but the tranfer procedure when I stained the membranes with Coommassei Blue. There were no proteins on the membranes but molecular marker.

Here is the the 10X Tranfer buffer:

0.25 M Trizma Base
1.92 M G Glycine (MW 75.07)

For the 1X transfer buffer, 20% Methanol was added.

Transfer procedure:
100 V for 1 hr with the cold pack. PVDF membrane wet for 30 min in Methano before transferring.
I am looking for help from all of you. Thanks a lot.

Yi

#2 Sprag

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Posted 14 January 2005 - 11:38 AM

I would assume there's something wrong with your sample prep, as your transfer seems to work according to the markers; if the marker is there, the protein (if any) has been transfered.

try to measure the protein concentration in you sample(s) before you run your gel.

good luck

ps..There's nothing wrong with your transfer buffer...

#3 thefallguy

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Posted 18 January 2005 - 08:02 AM

Claudin-5 needs higher transfer time than usual. Triple the time in your procedure, reduce buffer methanol content to 5%

#4 yyang

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Posted 20 January 2005 - 02:23 PM

Big thanks to Sprag and Thefallguy for your helps.

Best regards,

Yi

#5 badcell

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Posted 21 January 2005 - 10:53 AM

Hi, yyang. Maybe is just a typo, but you say that you wet the membrane in methanol for 30 min. I assume you mean 30 sec. Right?
Have you stained with coomassie the gel after the transfer to see if the proteins are still there? Sometimes you can get a complete transfer of the markers but just a very partial transfer of your proteins, as markers have very little quantity of protein, you only see them becase they are dyed. If the proteins are still in the gel, then maybe is a problem with your buffer (pH?); if the proteins are neither in the gel nor the membrane, then the problem is in your membrane- the proteins are getting out of the gel but for some reason they are not sticking to the membrane. Hope it helps! Cheers.
Science is a wonderful thing if one does not have to earn one's living at it
(A.Einstein)

#6 luminb

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Posted 23 January 2005 - 05:53 PM

you didn't mention that if there is any proteins left on your gel or not. also, when you said that the samples did not get transferred, you didn't specify if there was any commasie staining on your membrane at all.
if the transfer time is not sufficient, you will still see lots of proteins on the membrane because some proteins with small mw will still get transferred provided that you used 100v, 1hr.
I won't say it is the problem with your membrane as pvdf has small pore size not to let go proteins. in addition, your marker got transferred. i assume that your marker was completely transferred (if the dye is all on the membrane but no dye in your gel).
so please check commassie blue on your gel, if they are all still in the gel, then check the condition of your samples. protein concentration, lysis buffer, sds loading buffer, per se. prolonging the transferring time won't do too good, given that 100v, 1h should be good enough for proteins <120 kd.

good luck




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