Author Topic: high MW protein still remain in the gel?  (Read 6843 times)

andye

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high MW protein still remain in the gel?
« on: August 22, 2006, 05:00:00 PM »
why prestained marker protein transfered to the NC membrance but  high MW protein were still remained in the gel?
I have pre-wet my gel (10%) and Hybound membrance in transfer buffer (including 20% MeOH), and transfer in the refrigerator (4C) in 2 hours.
when I finished the transfering, I have found that  the prestained were transered to membrance except of 200Mr protein, and the membrance and gel seemed drying. when I stained my gel, I found a lot of protein were still remained in the gel except of low MW proteins (<40).  
transfer time is not enough? who can tell me the reasons?
Thanks!

Sjouke Hoving

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Re: high MW protein still remain in the gel? (andye)
« Reply #1 on: August 23, 2006, 05:00:00 PM »
In general, high MW protein transfer not very efficient. It is a limitation of the blotting technology.

sjouke


sofiaedlund

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Re: high MW protein still remain in the gel? (andye)
« Reply #2 on: September 03, 2006, 05:00:00 PM »
Dear Andye

Just some comments:
I agree on what "sjouke" said. It is always more hard to get HW proteins to be transferred. It also depends on which transfer system you use. For high molecular weight proteins a tank (wet) transfer is to be preferred. Then also this keeps the gel and membrane wet the whole transfer time and you will get more even transfer. It is really important to try out the transfer time for your protein. As you did with the staining of the gel is good to do to see how much that got transferred. You can also put two membranes on top of each other and run different transfer times and see when you protein is transferred from one membrane to the other. Then you will see the stop time. I think that two hours could be too short. Try out different transfer time and different V or Am. Still it is important to have the marker side by side, just to see that the transfer was OK in the aspect of direction. Still the time for the more big proteins would be different to the marker, since not all markers use very high molecular weight proteins.

Best Regards
Sofia E


mandar-seo

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Re: high MW protein still remain in the gel? (sjouke)
« Reply #3 on: October 18, 2006, 05:00:00 PM »
Great information!

With regards,
Mandar Thosar


mkvarshney

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Re: high MW protein still remain in the gel?
« Reply #4 on: July 10, 2011, 07:19:25 AM »
Hi,
For efficient transfer of HW proteins you may add 0.05% SDS to your transfer buffer. It will aid in transfer of HW proteins but remember that you may not get LW proteins due to over transfer of them because of extra charge by SDS. This is just for HW proteins...

Sjouke Hoving

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Re: high MW protein still remain in the gel?
« Reply #5 on: October 20, 2011, 02:41:18 AM »
How large is your protein of interest?

I was successful with blotting a 500 kDa protein, using a (mini BioRad) wet-blotting system (25 mM Tris, 192 mM Glycine, 5% MeOH, pH 8.3 - don't adjust pH). Conditions: PVDF membrane, 100 V for 2 hrs. The whole set-up is placed in an ice bucket.

sjouke