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Pippuri
Member since: Jul 12, 2009
From: Georgia, United States
Status: Protein Chemistry & Glycobiology Moderator
My points: 1053    what's this
Name: [Privacy]
 


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Jan 24, 2012    11:20 AM

Replied to topic in Protein Chemistry forum   Protein purification from inclusion body in the cell pellet

Hi Kritika, Yes, lowering the culture temperature also requires longer time before the culture is ready to be harvested, For the pH of your lysis buffer, it depends on the pI of your protein- keep in mind that when the pH is very close/equal to the pI of your protein, the protein tends to form ...

Jan 23, 2012    05:21 AM

Replied to topic in Protein Chemistry forum   Protein purification from inclusion body in the cell pellet

Hi Kritika, You can try to culture at lower temperature, says, 16 or 25 degree. Some of the proteins will stay in their soluble forms at these temperature. Also, I'd like to make sure I understood correctly- even with the presence of guanidine, your protein still remain in the pellet? Good ...

Jan 21, 2012    07:23 AM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Wrong protein sizes in western

Hi JoanaC, In both cases, are those extracellular proteins? Have they been shown to be glycosylated? Often, heavyly glycosyated protein will show a severe retention in SDS-PAGE. Cheers.

Jan 18, 2012    07:35 AM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   poor tranfer below ~25kDa

Hi Cecilia, One thing that you can definitely try is to change the membrane from pore size 0.45 um to 0.22 um. The only thing is with a 0.22 um membrane, you need to wash more extensive in order to eliminate background for it yields higher background in general. Good luck.

Jan 1, 2012    10:32 AM

Replied to topic in Cloning (Recombinant Gene Expression) forum   problem in protein expression

Hi dalim2000, When you mentioned you couldn't express the clone with the extra +ive charge AA, what kind of samples you were looking at? Total lysate or S/N? And what kind of conditions you have tried so far??

Nov 27, 2011    03:03 PM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Western Blotting of Proteins around 10 kDa

Hi scichem, For a typical tank transfer apparattus from Bio-Rad, such as the Criterion Blotter that you are using, it should be able to handle voltage up to 100 V- If you aren't able to run higher than 35 V something must have gone wrong! One reason may be the whole system is overheated. One solu ...

Nov 27, 2011    02:50 PM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Uneven transfer across Western Blot

Hi mcorso10, If the sponges that you used for making the sandwich is getting very thin, you may want to invest in new sponges. Sponges that are too thin may cause imperfect transferring, if other possibilities have all been checked out. Good luck.

Nov 7, 2011    08:48 AM

Replied to topic in Antibody Based Technologies forum   How to separate A/G beads from the attached antibodies?

Hi viralscience, One way to do it is to incubate with 0.1 M glycine (pH2.5 to 3) at ambient temperature for 5 to 10 min. However, neutralization of your sample (typically, with 1/10 volume of 1 M Tris, pH 8) immediately after acidic elution is needed. Good luck.

Oct 28, 2011    09:05 AM

Replied to topic in Microbiology forum   comparing protiens at the surface

Hi Nazunderwent, I might be hand-waving here, but one of the approaches comes across my mind is outlined as follow- (1) Biotinylated surface proteins with NHS chemistry (2) Strepavidin (or avidin etc) pull down (3) Mass-spec ID Good luck!

Oct 27, 2011    06:02 AM

Replied to topic in Cell Culture and Tissue Culture forum   3T3-L1 differentiation

Hi AGlabswed, I got mine from Roche. Cat # 11 376 497 001. Cheers!

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