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Pippuri
Member since: Jul 12, 2009
From: Georgia, United States
Status: Protein Chemistry & Glycobiology Moderator
My points: 1047    what's this
Name: [Privacy]
 


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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!

Oct 19, 2011    01:47 PM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Pull down assay

HI karkan, EDTA is "optional" for lysis buffer. I prefer to have EDTA in my lysis buffer though, as it can inhibit some metal-dependent proteases... If you are getting positive result, I don't think it is much a cencern really. Good luck.

Oct 19, 2011    01:34 PM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Pull down assay

Hi karkan, N = NaCl E = EDTA T = Tris N = NP40 Since TBS is basically Tris and NaCl, if you have EDTA and NP40 in your recipe, I think they are pretty much the same thing. Good luck!

Oct 17, 2011    06:56 AM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Good crosslinker for co-IP

Hi imortalspirit, To cross-link Ab to Protein A/G beads, I routinely use DSS (Disuccinimidyl suberate). There is a more expensive but more stable derivative, called BS3 [Bis(sulfosuccinimidyl) suberate] will do too. While I have no experience for in vivo crosslinking of protein complexes, but ...

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