Western Blot on an IEF separated recombinant protein - (May/10/2006 )

Hi All,

I was just wondering has anyone carried out a Western Blot (using Mouse Anti-His to probe) on an IEF gel?

I have tried several times with Novex Vertical IEF gels, however, no signal is noted in the Western. Currently I use semi-dry transfer.

Any comments / suggestions would be great.

Thanks in advance....

B.

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(stupid question) do you equilibrate your gel in transfer buffer?

if you don't then your proteins will be neutral (uncharged) because they are sitting at their pI.

if you do, ief gels are macroporous so that they are non-restrictive, you may have washed the protein out of the gel.

you may be able to equilibrate for a shorter time.

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Hey "The Elder",

Thanks for your reply.

Yes I soaked in transfer buffer, but I just carried out a 15mins soak.

I'll try a shorter soak.



Thanks again .

B.

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A stupider question: Are you sure you´re blotting the correct way? When you use IEF gels you should blot the opposite way, i.e. the membrane under the gel. At least this is how it is done in invitrogens system

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Hi Kottila,


Thanks for your post. I'm not sure if I totally understand your suggestion.

Heres how I normally blot (see pdf)...is this the same as your method?

Thanks again,

B.

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That's the same way as I do it, so that's not it. Are you using some kind of indicator to check if the blotting worked? (a ladder that is visible on the membrane or some color that is transfered from the gel to membrane)?

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hello,

a few thoughts for you...

- have you blotted your recombinant protein onto PVDF from a traditional 1D SDS-PAGE? if not, how do you know that the reason you're not getting signal from your IEF strip isn't due to the fact that you have bad antibody? anti-his Abs are notoriously crappy (despite what the manufacturers might say), and usually should be used only if necessary. if your anti-his Ab gives you signal from a 1D western and not the 2D, then it's possible your protein might be precipitating (and therefore, not transferring to your PVDF) in the IEF gel. it wouldn't be the first time someone has lost a protein in an IEF gel.

- if you are worried about your transfer, have you stained your PVDF to see just how much protein is trasferring from your IEF strip. if you're having poor trasfer issues, this would be a way to test for that.

- another thing, have you tried a contact blot? i'm not sure how well your protein is being expressed (is it in E. coli, or a mamalian system?), but if it's being expressed at a pretty high level, you can probably get away with taking your strip, and pressing it directly onto wetted PVDF membrane (remove the methanol, and wash w/ water) for about 10 minutes. do this on top of a wet piece of blotting paper to make sure your membrane doesn't dry prematurely. after 10 min, let your membrane dry for 1/2 hr at room temp. re-wet it w/ methanol, block it as you normally would after a western, and probe. this is about as low-tech as you can get for blotting, but it's quick and works well for proteins that are highly expressed. if you protein isn't highly expressed, don't waste your time with this technique.

good luck,

jon

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