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Pippuri
Member since: Jul 12, 2009
From: Georgia, United States
Status: Protein Chemistry & Glycobiology Moderator
My points: 1081    what's this
Name: [Privacy]
 


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Sep 7, 2012    03:40 PM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Resolving two proteins of very close size

Hi Sam's Lab, If you run the sample on a 10% gel longer than you usually run so that ~35kDa region hits the bottom of the gel, you shoule be able to get those two to separate. Good luck

Jul 22, 2012    07:57 AM

Replied to topic in Protein Chemistry forum   Membrane protein quantification

Hi hermione, Two questions: 1) What kind of buffer is your membrane proteins stored in? 2) What kind of kit you are using right now?

Jul 5, 2012    05:50 AM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Far Western blot question

Hi Klof, It depends on the interaction between your proteins, the gel can be run with native or denature condition, although most far western was done under native condition. When I read back my previous reply, I might have messed up with X/Y- Anyway, the control should be depleted of your tar ...

Jul 3, 2012    06:59 AM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Far Western blot question

Hi Klof, You need to include control that has been depleted of your protein Y to address such issue.Two ways of doing this are- (1) if you have an antibody against protein Y, then use that Ab Y-depleted lysate as your control (2) if you can get a knockout/knockdown line of protein Y, then use t ...

Jun 26, 2012    07:21 AM

Replied to topic in Glycobiology forum   Recommendations for suppliers of exoglycosidases wanted

Hi SWilliams, Try either ProZyme or New England Biolabs. It usually takes SUPER long time to get decent cleavage (like 48 hours). Adding more enzyme after the first 24 hours incubation will help. Good luck.

Jun 21, 2012    01:09 PM

Replied to topic in Antibody Based Technologies forum   Affinity Purification of Chicken Antibodies

Hi Joanna_LaBresh, if your IgY is captured on the protein column, low pH should be able to elute the IgY VERY efficient. Key is you have to incubate the column in such condition for ~5 min before you start collecting the eluate. If you have already done that, then the question is, have you follow ...

Jun 21, 2012    12:33 PM

Replied to topic in Antibody Based Technologies forum   Affinity Purification of Chicken Antibodies

Hi Joanna_LaBresh, Can you provide the condition(s) that you have tried? It is easier to troubleshoot if this information is available. Cheers.

Apr 26, 2012    06:44 AM

Replied to topic in Biochemistry forum   DNA pull down strategy to purify a transcriptional regulator

Hi adlheras, Threorically, I don't see any problem using this approach to enrich for your transcription factor of interested. There are a few conditions you can optimize- (1) You can preclear your sample with Streptavidin beads alone before the pull down experiment to eliminate any potenti ...

Apr 25, 2012    07:46 AM

Replied to topic in Biochemistry forum   DNA pull down strategy to purify a transcriptional regulator

Hi adlheras, Two questions; (1) Is the transcription factor of your interested shown as the major component on the coomassie stained gel regardless the presence of other proteins? (2) Can you please provide the full recipe of your binding/washing condition? Cheers.

Apr 24, 2012    09:36 AM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Large Protein Western Blot

Hi Suuny5188, Yes, as the transmembrane hydrophobic regions tend to associate which leads to the formation aggregates once the temperature drop to ambient temperature. Try to us 60 to 70 deg for 5 to 15 min instead. Good luck.

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