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Pippuri
Member since: Jul 12, 2009
From: Georgia, United States
Status: Protein Chemistry & Glycobiology Moderator
My points: 1069    what's this
Name: [Privacy]
 


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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.

Apr 24, 2012    09:00 AM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Western blot transfer inefficient

Hi Christina86, Keep in mind that one can't directly compare the staining intensity on the gel and on the membrane, as the staining efficiency aren't equal between these two materials. If you are extremely cautious, you can do a test by running identical samples on 2 gels, one is stained d ...

Apr 24, 2012    07:13 AM

Replied to topic in Cloning (Recombinant Gene Expression) forum   Calcium phosphate transfection problem

Hi imortalspirit, If the white precipitate is form after mixing the plasmid with the other components which make the solution looks a little cloudy, I don't think it will affect your transfection efficiency (speaking of which, do you have a control plasmid like GFP to keep track of your transfect ...

Apr 24, 2012    06:39 AM

Hi yanglin0, Have you also included secondary antibody alone? It could be that the condition of the fluorescent conjugated secondary Ab is not optimal. Good luck.

Apr 24, 2012    06:35 AM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Western blot transfer inefficient

Hi Christina86, Were you able to detect your protein on the membrane at all? Based on your prestained marker, I bet you also have decent amount of protein on the membrane regardless residual proteins left on the gel. In my opinion, it depends on how much protein per lane you loaded. Too mu ...

Apr 12, 2012    06:35 AM

Replied to topic in Biochemistry forum   Mechanism of lowering of blood glucose

Hi Sambos, You can find assays for all your enzyme of interested in the following link- Enzyme Explorer/Sigma-Aldrich Good luck.

Mar 29, 2012    09:24 AM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Western blot mystery - possibly a sample preparation problem?

Hi Mark, I have not used the method you mentioned to prepare sample for WB, so it is pretty hard to tell how it might go wrong. However, I routinely use RIPA buffer to lyse cells and an electronic homoginizer to aid protein extraction from tissue samples as depicted in this link (from Abcam). Al ...

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