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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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Jul 21, 2014    09:54 PM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Problem with SDS-PAGE

Hi kola, One possible explanation leads to such staining pattern is bacterial growth in the running buffer. Try to prepare fresh running buffer from scratch and store it in a fresh container and run a gel again. Good luck

Jul 18, 2014    07:08 PM

Replied to topic in Biochemistry forum   western blotting

Hi milsey, Try to incubate your sample at 56 to 70 degree for 15 min instead of 95 to 100 degree often help with keeping membrane proteins in solution and may help with your detection. Good luck

Jul 17, 2014    12:11 AM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   yield of purification by IP and Co-IP

Hi dorvashmr, One option, which can definitely get you a lot more proteins than IP is to get a plasmid that carry your protein of interest, overexpress it in E. coli and perform a quick affinity purification. It may take longer time (having to do transformation, grow the bugs and finally purific ...

Jul 12, 2014    12:36 AM

Replied to topic in Biochemistry forum   WESTERN BLOT - high molecular weight protein

Hi ndora, If you only loaded 100ng of cortical and hippocampal lysates per lane, I suggest you to go higher amount than that. Says, 20 to 30ug. Also, for transferring a protein that large, you may want to transfer for longer time- 20 to 30 V for 16 hours at 4 degree. Another thing is to dro ...

Jun 12, 2014    04:26 PM

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

Hi Mi: Yes, if you perform the electrophoresis on a 20% SDS-PAGE. Good luck

May 13, 2014    04:25 PM

Replied to topic in Cell Culture and Tissue Culture forum   determining GLUT4 to the cell membrane bound

Hi lunaoreaD, For muscle cells and adipocytes, both of which express GLUT4 endogenously, you do not need to overexpress the transporter, given that you have the proper antibody to detect the endogenous form of GLUT4. Good luck.

Apr 26, 2014    06:01 PM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   LAEMMLI BUFFER MISTAKE

maria258, As long as the final pH of the Tris buffer is 6.8, it doesn't really matter whether you started with Tris.HCl or Tris.base. Your samples are absolutely fine. Good luck.

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 ...

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