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Pippuri
Member since: Jul 12, 2009
From: Georgia, United States
Status: Protein Chemistry & Glycobiology Moderator
My points: 1033    what's this
Name: [Privacy]
 


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Apr 17, 2014    10:54 PM

Replied to topic in Protein Chemistry forum   Bradford calculation problem

Hi anuranjani2014, 1) yes 2) yes 3) that is 1/10 dilution, thus it has to be multiple 10X 4) yes Good luck.

Apr 15, 2014    07:32 PM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Interesting Immunoprecipitation Problem

Dncrwlye, It is an interesting problem instest. Can you please upload all your gels/WB results as you mentioned? Thanks.

Mar 27, 2014    02:26 PM

Hi shubhash chaudhary, How much is the mass shift? Does it correspond to the Mw of GFP? What kind of tranfection efficiency you are getting? If the shift matches GFP Mw and the transfection efficiency isn't that high, an obvious explanation is that, the WB on whole cell lysate is picking up end ...

Mar 19, 2014    07:34 PM

Replied to topic in Cell Culture and Tissue Culture forum   Dissolving drug in DMSO for use with cells

Hi migwke, No, it is impossible to get the DMSO below 0.1% v/v. Your calculation is absolutely correct. Cheers.

Feb 27, 2014    12:26 AM

Replied to topic in Cloning (Recombinant Gene Expression) forum   pLenti6.3/TO/V5-DEST

Hi kphd, I have not used pLenti6.3, but have used pLenti4 before. Don't they also provide a construct carrying LacZ along with your kit? Otherwise, you can clone GFP into the destination vector and use it as your control... Cheers.

Feb 27, 2014    12:22 AM

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

Hi cagr, Are you using a tank(wet) transfer apparattus? Is it a PVDF or nitrocellulose membrane?

Feb 18, 2014    08:49 PM

Replied to topic in Cell Signaling/Transduction forum   Exosome isolation problems

Hi jseibert, My experience is (1) to pipet VERY slow/gently; (2) not to let the samples sit "idly" after the centrifugation (i.e. process quickily right after the centrifuge stops); (3) yes you are right, use the smaller pipet... Good luck!

Feb 11, 2014    06:06 PM

Replied to topic in Protein Chemistry forum   Protein Induction with IPTG

Hi ratno, Threorically, yes-- but at your own risk of having protein degradation. (hence I won't recommend anyone to do that) That being said, the best way is to spin down the bugs- Once you have the pellet, you can store the pellet in the freezer (-20degC or -80degC) until the next step. G ...

Feb 7, 2014    04:40 AM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Ghost Bands in Western Blotting?

Hi samwich, Getting ghost bands in WB is usually associated with too much of protein and/or antibody (espcially primary antibody). If you are getting ghost bands, it is very doubtful that the conditions were indeed optimized- It could be "suboptimized", in a sense that, you are getting ...

Jan 31, 2014    08:16 PM

Replied to topic in Neuroscience forum   Protein Extraction from cell culture media

Hi Tamsinash, I think you do need to try different conditions and see how your cells look. You should not remove the serum during the routine culturing/maintaining/plating steps, but do get rid of the serum while setting up your experiments. Whether the cell will be deprived of nutrients/growt ...

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