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Incomplete transfer


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#1 mohsen

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Posted 17 May 2011 - 05:06 AM

Hi everybody,

I am trying to transfer some cell lysate to NC membrane. I use 1.5 mm minigels and then transfer in towbin buffer, pH 8.3, containing 20% mehtanol in maximum current (400 mA, 150-120 volts) and also use the cooling unit, for 1.5 h.
The problem is that I can not transfer the bands well. I use the Colorburst Sigma marker and it's transfered well and nothing's left in the gel, so it looks everything's alright. I can see the bands on the membrane using ponceau S. But then when I stain the gel with Coomassie Blue, still there's a lot of protein left in the gel.
I tried increasing the transfer time to 2 hours, and also put a second NC membrane behind the first one to check if the bands would pass through the first membrane. I can see some bands have passed, so it looks like the transfer time was too long, but still I can see a lot of protein left in the gel.

Any help would be appreciated.

Cheers
Mohsen

#2 mdfenko

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Posted 17 May 2011 - 07:21 AM

are you seeing only high molecular weight proteins left in the gel? this is normal. larger proteins almost never transfer completely.

if you want to transfer longer then you can use a smaller pore membrane (0.2um).

you can also add up to 0.05% sds to the transfer buffer to facilitate transfer of large proteins (do not change the methanol concentration).
talent does what it can
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#3 mohsen

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Posted 17 May 2011 - 08:12 AM

well I see the whole spectrum of sizes, from 20 kD to 200 kD. It looks like the gel has not been exposed to transfer at all! And I'm looking for a protein of normal size, about 70 kD.

Thanks for comment.

#4 mohsen

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Posted 18 May 2011 - 12:37 AM

Please help.

#5 mdfenko

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Posted 19 May 2011 - 07:16 AM

try adding 0.05% sds to the transfer buffer.
talent does what it can
genius does what it must
i do what i get paid to do

#6 mohsen

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Posted 20 May 2011 - 01:43 AM

@mdfenko: Thank you so much for help.

By the way I tried another buffer, 10 mM CAPS, pH=11, 10% methanol and it works fine. But I will try adding 0.05% SDS to Towbin buffer as well.

And a question: I'm going to purchase PVDF membrane, since I will need stripping the membrane, and they say PVDF would retain the bound protein better than NC. Have you ever tried the Hybond LFP (amersham's PVDF)ever? On the website of GE Healthcare it's stated that it will produce low background for florescent development (ECL). Which brand do you recommend? I have compared the NC of thermo scientific and Amersham Hybond and the Hybond looks to retain the bound protein much better, judged based on the pre-stained marker. So maybe the PVDF Hybond would work fine as well.

Many thanks in advanced

#7 mdfenko

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Posted 20 May 2011 - 11:12 AM

for westerns i only used supported nitrocellulose and i didn't strip and reprobe (i was depositing color).

i used pvdf for peptide sequencing. it was a special(?) grade sold by the machine's manufacturer (i wasn't given a choice).

some membrane manufacturers make a "fluorescence grade" membrane (as you saw from ge healthcare). you can request samples to evaluate.
talent does what it can
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i do what i get paid to do

#8 mohsen

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Posted 21 May 2011 - 09:10 AM

Thanks. I will ask them for some trial samples.




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