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Pippuri
Member since: Jul 12, 2009
From: Georgia, United States
Status: Protein Chemistry & Glycobiology Moderator
My points: 1031    what's this
Name: [Privacy]
 


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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 ...

Oct 11, 2011    08:23 AM

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

Hi imortalspirit, Are you looking for a crosslinker that crosslink your antibody to the beads or a crosslinker that crosslink for protein complexex? I personally like the crosslinker collection from Pierce/Thermo Fisher. You may want to go through their website and pick one that suit your exp ...

Oct 7, 2011    09:16 AM

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

Hi tisana86, Yes, I do believe using 90V instead of 30V can compesate for the shorter transfering time. Good luck!

Oct 6, 2011    07:07 AM

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

Hi tisana86, What % of SDS-PAGE did you use? I suggest a gradient gel-- 4-15% should do fine. The other thing, I am not sure what kind of system you are using, but for a wet transfer system, I usually use 90V, 1.5 hours at 4 deg, whereas for a semi-dry transfer, I use 20 V 30 to 60 min. (Both ...

Oct 4, 2011    07:38 AM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   recombinant protein detection

Hi Afshari, I think you loaded too much sample, which led to substrate depletion (the fact that you can see "a thick and sharp band " on SDS-PAGE is a dead on indication). To overcome this problem, you have to load a lot less sample per lane- I suggest you a dilution of 1/500 as a st ...

Sep 22, 2011    06:23 AM

Replied to topic in Cell Culture and Tissue Culture forum   bovine insulin

Hi biolyse21, If you are looking for insulin used in adipocyte cultures, I routinely use human (instead of bovine) insulin from Roche. Good luck!

Sep 15, 2011    09:41 AM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Isolation of large membrane proteins and western blotting

Hi Peter, Can you be more specific about "not successful"? You did not see the difference that you expected, or you couldn't detect the protein of interest at all, or the biotinylation was not efficient, or the avidin affinity pull down didn't yield anything?? It will be a great h ...

Sep 3, 2011    03:17 PM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Transfection of HEK cells

Hi swati9, It still soulds like your problems would be either transfection or immunoblotting steps, or both. To tease out/pinpoint the real culprit, I suggest you to- (1) use a GFP control plasmid in parallel to confirm the transfection efficiency (2) optimize western blot conditions ...

Aug 29, 2011    12:56 PM

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

Hi Shahid Mohd, Yes, you can use human insulin instead of bovine insulin- I used human insulin for such purpose indeed. Good luck.

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