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#1 eyes

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Posted 11 February 2011 - 07:23 AM

A very stupid question for one like me who is starting in protein studies.
If I choose the wrong acrylamide concentration for my SDS-PAGE is it possible that I don't get my protein after incubating with my antibody?
In other words it is clear for me that a good separation is important to be sure that the protein I see on my blot is that I'm looking for, but do you think if I'm running a 18% gel and therefore the proteins do not separate well, it could be possible that I don't detect the one I'm interested in?
THanks in advacne
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#2 knuf

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Posted 11 February 2011 - 11:41 AM

I would think that this would only be a problem if you use a concentration that is too low and you accidently run the protein off the gel. On the other hand, I don't know how high is too high for the protein to enter the gel. Assuming that the protein is in the gel and was transferred to the membrane, though, I would think that the gel concentration would only affect the resolution of the band, and not the actual detection, however I have not run an 18% gel, so I really don't know for sure.

#3 bob1

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Posted 11 February 2011 - 09:26 PM

If the gel is too high a concentration the larger proteins will not migrate onto the gel at all, so you may not see them on the transfer, depending on the size of the proteins you are looking for.

Also, see attached document for the concentrations to use for effective separation (Roche Lab FAQs, pg 92).

Attached Files



#4 eyes

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Posted 14 February 2011 - 05:43 AM

Ok thanks for your replays.
The problem that I have is related to the detection of small proteins (about 4 and 17 KDa) that I'm not sure are present in my sample and that so far I was not able to detect using a standard western method. I checked the forum and I found some suggestions to follow but I wanted just to understand if the run could be the cause of my failures.
Thanks again

#5 bob1

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Posted 14 February 2011 - 02:55 PM

With proteins of that size it is very likely that they are blowing through the membrane on transfer - what membrane type are you using and how long are you transferring for?

#6 eyes

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Posted 15 February 2011 - 03:41 AM

With proteins of that size it is very likely that they are blowing through the membrane on transfer - what membrane type are you using and how long are you transferring for?


I started working with a 45 um nitrocellulose membrane and I blotted for about 30' at 100 V (wet). Reading the forum I'm pretty sure this are not the right conditions. Which do you think should be the best?
Thanks

#7 bob1

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Posted 15 February 2011 - 01:13 PM

It will be tricky, you will probably need to use PVDF membrane and blot for a shorter time at a lower voltage, but you will need to determine the actual times and conditions by yourself as they depend on the conditions and equipment you are using.

#8 mdfenko

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Posted 15 February 2011 - 01:26 PM

i would not use a pore larger than 0.22um for low molecular weight proteins and peptides (i use 0.2um pore supported nitrocellulose).

i would also use a tris-tricine gel instead of tris-glycine. it is better for separating low molecular weight proteins and peptides.

Edited by mdfenko, 15 February 2011 - 01:27 PM.

talent does what it can
genius does what it must
i do what i get paid to do

#9 sciencerage

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Posted 17 February 2011 - 03:19 PM

I second the advice above - you definitely need a membrane with a smaller pore (I also use 0.2um nitrocellulose) and tricine-SDS-PAGE will give you much better separation of very small proteins. You can check out this paper for some advice and a reagent recipes - PMID: 17406207. Another pointer for small proteins is to leave SDS out of your transfer buffer - it can hinder the binding of small proteins. Good luck!




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