western blotting

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shylet
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western blotting

I am trying to do a western blot to detect the levels of expression of a large protein made up of 5 subunits 140 KDa for each subuit. I am getting signal at the position of the wells, so the protein is trapped in the wells. I use a loading buffer which has Beta-mercaptoethanol, which should reduce the protein to where I can get the subusits separate into monomers but for some reason that is not happening. Please help? What can I do differently?

DD
DD's picture
shylet wrote:I am trying to

shylet wrote:

I am trying to do a western blot to detect the levels of expression of a large protein made up of 5 subunits 140 KDa for each subuit. I am getting signal at the position of the wells, so the protein is trapped in the wells. I use a loading buffer which has Beta-mercaptoethanol, which should reduce the protein to where I can get the subusits separate into monomers but for some reason that is not happening. Please help? What can I do differently?

Before I can give an answer I need to know what kind of gel and running buffer are you using. The protein has to be solubilized before we add it to the loading gel, boiling might help too.

shylet
shylet's picture
I am using SDS Bis Tris

I am using SDS Bis Tris glycine buffer and Bis glycine gel

Soudabeh
Soudabeh's picture
shylet wrote:I am using SDS

shylet wrote:

I am using SDS Bis Tris glycine buffer and Bis glycine gel[/
omid
omid's picture
shylet wrote:I am using SDS

shylet wrote:

I am using SDS Bis Tris glycine buffer and Bis glycine gel

Try to use 4-12% gel which can help to separate the big protein.

Fraser Moss
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I suspect, you protein is a

I suspect, you protein is a trasmembrane protein form the way you describe it. When I was doing my PhD I had a similar experience with a much smaller membrane protein the was not supposed to form a multimer but aggregated in the wells of the gel.

You have not said if you boil your samples or not before loading. In my experience, transmembrane proteins really DO NOT like being boiled before you load them on the gel. You need to rely on the SDS and the mercapto to do the work. I have run voltage gated sodium and calcium channel alpha subunits on gels this way with great success. This did not 100% cure the well aggregation in my smaller protein but enough got into the gel to be detected. You can also try increasing your SDS concentration in your running buffer.

Another Really good idea that significantly reduced the aggregation in my situation was to try PAGE UREA gels. People use 4-7M Urea in their Gels and Buffer with good results. If you dont want to buy/make a load of Urea gels, you can pre run your gel in 7M urea buffer for a couple of hours to run it through the matrix.

Good luck.

karusci
karusci's picture
frasermoss wrote:I suspect,

frasermoss wrote:

I suspect, you protein is a trasmembrane protein form the way you describe it. When I was doing my PhD I had a similar experience with a much smaller membrane protein the was not supposed to form a multimer but aggregated in the wells of the gel.

You have not said if you boil your samples or not before loading. In my experience, transmembrane proteins really DO NOT like being boiled before you load them on the gel. You need to rely on the SDS and the mercapto to do the work. I have run voltage gated sodium and calcium channel alpha subunits on gels this way with great success. This did not 100% cure the well aggregation in my smaller protein but enough got into the gel to be detected. You can also try increasing your SDS concentration in your running buffer.

Another Really good idea that significantly reduced the aggregation in my situation was to try PAGE UREA gels. People use 4-7M Urea in their Gels and Buffer with good results. If you dont want to buy/make a load of Urea gels, you can pre run your gel in 7M urea buffer for a couple of hours to run it through the matrix.

Good luck.

Hi,
I just happened to read your posting, a member of my lab is working on purification of a membrane protein and we came across issues with protein solubility (insoluble) and related to SDS-PAGE run as you noted above. I think that we should give the membrane protein the right environment/condion, thus the hydrophobic condition such as micellar environment. I would like to get your opinion on this? and aslo If you have any good information on purification of membrane bound protein, and resolving solubility issues on this group of protein is very much appreciated.
thanks
Karu

Fraser Moss
Fraser Moss's picture
Well I think the micellar

Well I think the micellar idea is an interesting one.

I have not come across its application in a PAGE gel, but I know it is used in capilliary electrophoresis.

Maybe instead of an anionic sufactant like SDS you could consider something neutral like sodium cholate to get insoluble membrane proteins to solublize. You'd have to experiment with running buffers and makem sure all your standards and ladders were prepared the same as the samples as the micellar environment will no doubt effect mobility.

As far as my experience resolving membrane protein solubility, my initial post pretty much covered what I did to get my proteins to run. You can do immunoprecipitations to try and increase your signal on your gel too, but I reccomend trying to use a different antibody for IP to detection on the western blot where possible. However, working with less protein (at the lower end of the detection limit of your antbody) is probably advisable, as high local concentrations of your denatured protein in the running buffer is probably where you problems starts.

vanishing
vanishing's picture
DD wrote:DD wrote:Before I

DD wrote:

DD wrote:
Before I can give an answer I need to know what kind of gel and running buffer are you using. The protein has to be solubilized before we add it to the loading gel, boiling might help too.

I would not boil, that might lead to protein aggregates (especially with membrane proteins)

karusci
karusci's picture
Hi

Hi
Thanks for the information, We were using 4-20% Tris-Gly gel. As it is noted in this forum, we noticed that the boliling has an effect on the portein solubility in the loading buffer. Therefore, we do not boil sample (membrane bound protein prep) prior to loading onto the gel. My question was in general, as to any new development on the purification of membrane bound protein (MBP). It is known that MBPs are difficult to purify when compared to the cytosolic ones. Any attempt to solubilize this protein make it diificult in the chromatographic step (AEX, CEX or affinity). So, I would like to know if any of our readers know any latest development in this area of purifying membrane bound protein.
Thanks

luciotremolizzo
luciotremolizzo's picture
we got the same kind of

we got the same kind of problems trying to resolve a transmembrane transporter VGLUT1... when we don't boil it is better but the immunoreactivity is not any more so specific and you got to be very careful to run your II ab test.

milsey
milsey's picture
 Hello everyone,

 Hello everyone,

I am trying to detect the expression of a membrane protein (170kDa) through western blotting. But I am having problem detecting this protein band after several attempts and I happened to come across this post which might help me. 

I am trying to overexpress this membrane protein and have added a tag sequence for detection convenient. Initially, I added Flag sequence but I couldn't detect this protein by using Flag antibody. Then I am thinking that this Flag may not be suitable for my protein (trapped in the protein structure) and therefore changed to HA and Myc tag, but still was not able to be detected. My problem is that I boiled my sample at 95 for 10 minutes before separating them on PAGE. Is it really not advisable to boil the sample before separation on a PAGE gel if my target is the membrane protein? Mind giving me some tips or letting me know a more detailed protocol to separate and detect membrane protein? Membrane purification technique is appreciated too.

Thanks

Chin Fen Teo
Chin Fen Teo's picture
Hi milsey,

Hi milsey,

Try to incubate your sample at 56 to 70 degree for 15 min instead of 95 to 100 degree often help with keeping membrane proteins in solution and may help with your detection.

Good luck