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vivlee's Content

There have been 6 items by vivlee (Search limited from 23-October 19)


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#144486 TFE in inducing alpha-helix formation in proteins

Posted by vivlee on 01 November 2012 - 05:58 PM in Protein and Proteomics

Shorter peptides are less likely to exhibit secondary structure. Secondary structure prediction programs often require at least 20AA peptides to predict any secondary structure. Short peptides tend to have unordered structure and this could be why you observed alpha-helical formation in the 32mer


Not even with the addition of TFE will it show secondary structure? I found that low temperature has some minor effect, but still more apparent as random coiling. Thanks for your help nonetheless!



#144362 How to avoid gel breakage after electrophoresis

Posted by vivlee on 30 October 2012 - 09:44 PM in SDS-PAGE and Western Blotting

Ooh, for imaging, I'll usually have it in pretty deep water/liquid in my tray, then I'll either try to get both (gloved) hands under the edges to lift it out all at once or I'll tease up an edge with some filter forceps, then lift it enough with one hand to get the other hand under it. That way the force of moving it is more distributed and less likely to tear. I wet my gloves before handling gels to help prevent sticking (you should be able to easily slide your finger over the gel without it grabbing).

As for on the imager, I'll usually coat the surface with water first, then set the gel in that one edge first, then easing it down to prevent bubbles.

For getting your gel off the plates, you could also try holding the plate over your container, then teasing a little piece of edge away, then doing the water bottle trick.


Ok, so i see that the water bottle trick is the key in any transferring. Thanks for the tips! And Happy Halloween! :)



#144357 How to avoid gel breakage after electrophoresis

Posted by vivlee on 30 October 2012 - 07:53 PM in SDS-PAGE and Western Blotting

One thing you could try is using a water squirt bottle to soak the gel while it's on one of the plates until an edge lifts up, then trying to use the water stream to kind of wedge the gel into your container for imaging. Or just put the whole plate in with your gel, which might soften up how strongly the gel sticks to your plate, then gently teasing the gel off with a spatula/forceps after incubating/rocking for a bit.

I'm assuming you're using your own gels on glass plates?


Any tip for removing the gel from water in a container onto the platform in digital imaging instrument without tearing it ?



#144354 How to avoid gel breakage after electrophoresis

Posted by vivlee on 30 October 2012 - 07:38 PM in SDS-PAGE and Western Blotting

Thanks! I will try loosening the gel edge first with water stream and rock the plate and gel together in water within a container.
And yes, I am using my own gels on glass plates, not commercial gels.



#144345 TFE in inducing alpha-helix formation in proteins

Posted by vivlee on 30 October 2012 - 05:24 PM in Protein and Proteomics

Hi all,

I have been doing TFE titraion on polypeptides at 4 degrees celcius using CD, and noticed its varying effects on different polypeptides. It was effective in inducing alpha-helical structure formation in one polypeptide but not another. One of the peptide I tested was a 16mer and the other is a 32mer, the 16mer is a part of the 32mer. Does the length of the peptide influence the effect of TFE, since TFE showed its effect on the 32mer but not the 16mer (even at 80%TFE)? Any other possible reason for the difference in TFE effects on the two mers I tested?

Thanks!



#144344 How to avoid gel breakage after electrophoresis

Posted by vivlee on 30 October 2012 - 05:14 PM in SDS-PAGE and Western Blotting

Hi all!
I have been running 8% SDS-gel (acrylamide) and the gel often breaks easily when I try to remove it from the glass plate after electrophoresis into a container for staining. I usually keep the gel wet during transferring into the container by letting deionized water running on the plate when I am trying to life the gel, though sometimes succussful, minor breakages still occur at times. Breakages also occurs at time when I need to take the gel out of the container for digital imaging after the staining.
Any SDS-PAGE experts out there that can help me minimize the chance of obtaining a broken gel? Thanks!




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