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quol's Content

There have been 8 items by quol (Search limited from 16-July 18)


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#11795 WGA coupling with CNBr-activated sepharose 4B

Posted by quol on 04 March 2005 - 06:06 AM in Protein and Proteomics

I used CNBr activated Sepharose 4B a lot and I had once the problem you described.
If the concentration of the ligand you want to couple is high, the handling must be carefull. Otherwise it aggregates.
To avoid such precipitates, I incubate the protein wiith Sepharose in the tube with gentle rotation end-over-end, having as little air as possible in the tube.
This should help



#8067 centrifuging

Posted by quol on 20 October 2004 - 11:42 PM in Protein and Proteomics

During sonication membranes break releasing the content of the cells and organells and they close again. No, you don't release transmembrane proteins, they will stay in the pellet.



#8066 Anybody knows any resolutions to improve TAP purification?

Posted by quol on 20 October 2004 - 11:38 PM in Protein and Proteomics

Can you specify the problem? which step you want to improve?



#6846 my bands spread as a line trough the gel

Posted by quol on 29 August 2004 - 11:11 PM in Protein and Proteomics

Maybe you overloaded the wells. Try to load less proteins. Of course the amount of protein for good separation depands on many factors as thickness of the gel, number of different proteins... but for example when using BioRad system (1mm gel 9x6 cm, 12 wells) I load not more than 30 mg of cellular extract- works perfectly.
Good luck



#6117 Coomassie blue staining

Posted by quol on 15 July 2004 - 11:29 PM in Protein and Proteomics

Hello Alexei,

In my experience I faced this problem many times. Generaly silver staining is indeed known to be more sensitive than Coomassie however it does not apply for all the proteins. For some proteins you can actually find the opposite.
Unfortunetely besides my own experience, I cannot give you any references.
Good luck :rolleyes:

Quol



#5880 Western Blotting background troubleshooting

Posted by quol on 22 June 2004 - 11:50 PM in SDS-PAGE and Western Blotting

Myself I never wash aways skim milk. I use 1% skim milk in TBST for bloking and then add primary antibodies. That reduces background.



#5841 SDS-PAGE running strangely

Posted by quol on 18 June 2004 - 05:29 AM in Protein and Proteomics

It depends on the acrylamide concentration. But genaraly the small proteins separate first. Your problem could be that the smallest proteins run out from the gel. Maybe you should run the electrophoresis shorter.



#5840 TCA precipitation of proteins

Posted by quol on 18 June 2004 - 05:24 AM in Protein and Proteomics

What is the mininal/optimal concentration of proteins for efficient precipitation with TCA? I lose my protein in the precipitation because of too low concentretion and I need to add some extra protein to the solution (I use BSA) but I don't want to overload the SDS gel.




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