Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum - Western Blot Forum http://www.molecularstation.com/forum/ Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures. en Thu, 15 Aug 2013 03:59:02 GMT vBulletin 60 http://www.molecularstation.com/forum/images/misc/rss.jpg Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum - Western Blot Forum http://www.molecularstation.com/forum/ downturned and upturned bands http://www.molecularstation.com/forum/western-blot-forum/88028-downturned-upturned-bands.html Fri, 02 Aug 2013 01:03:10 GMT I have the problem: Reviewers have asked me because there is an apparent discrepancy in band shape between DMT1 and actin. The DMT1 band is...
I have the problem: Reviewers have asked me because there is an apparent discrepancy in band shape between DMT1 and actin. The DMT1 band is downturned at the edges, but the actin band is upturned. I do not know to this effect should. Why is that?

Thanks
Giorgi Gisela
Argentina
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Western Blot Forum g.g http://www.molecularstation.com/forum/western-blot-forum/88028-downturned-upturned-bands.html
Sample preparation http://www.molecularstation.com/forum/western-blot-forum/88006-sample-preparation.html Sat, 20 Jul 2013 14:11:58 GMT Hello, Does anyone know if it is okay to freeze sample cells at -80C before lysis? The cells would be frozen, then taken out for lysis at a future...
Hello,

Does anyone know if it is okay to freeze sample cells at -80C before lysis? The cells would be frozen, then taken out for lysis at a future date.
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Western Blot Forum mtd0588 http://www.molecularstation.com/forum/western-blot-forum/88006-sample-preparation.html
Why would b-actin run differently using the same lysate but on a different gel? http://www.molecularstation.com/forum/western-blot-forum/88000-why-would-b-actin-run-differently-using-same-lysate-but-different-gel.html Wed, 17 Jul 2013 13:21:24 GMT I solubilized rat brain tissue using the Ambion PARIS kit according to their protocol. I ran 20ug of protein on a 10% gel, transferred to pvdf, etc....
I solubilized rat brain tissue using the Ambion PARIS kit according to their protocol. I ran 20ug of protein on a 10% gel, transferred to pvdf, etc. I first probed for phospho-jnk, then stripped the membrane using Abcam's recipe (SDS, Tris, BME), reprobed for total jnk, stripped again, and reprobed for b-actin (Thermofisher). No issues whatsoever. Great b-actin band in the right place, about 42kDa.

I then took the same lysates, load 20ug of protein onto each of 2 gels (12% this time), transferred to pvdf, etc., and then probed 1 for p-jnk and 1 for p-erk. I stripped them both, probed for total jnk and total erk, stripped again and probed both for b-actin (same dilution). This time b-actin shows up faintly around 42kDa but also has a much bolder band around 50-52kDa on both membranes. Can anyone tell me why this would happen? The only change was from a 10% to a 12% gel, as far as I know. I considered that perhaps there was ineffective stripping but that 50kDa band was not present previously on the ERK blots. Could something have gone wrong in the preparation for loading the samples? Can lysate taken from tissue rather than cells be unpredictable? I have used this same b-actin antibody before quite successfully with endothelial cells. Please advise. Thank you!
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Western Blot Forum pooderpops http://www.molecularstation.com/forum/western-blot-forum/88000-why-would-b-actin-run-differently-using-same-lysate-but-different-gel.html
antibodies of interest coming form the same host troubleshooting http://www.molecularstation.com/forum/western-blot-forum/87991-antibodies-interest-coming-form-same-host-troubleshooting.html Wed, 10 Jul 2013 14:37:18 GMT Hi all! I have a question and I really hope someone could help: I had one blot and wanted to check the expression of two different proteins on...
Hi all!

I have a question and I really hope someone could help:

I had one blot and wanted to check the expression of two different proteins on the same blot (mol weights are far from each other so no problem with that). So, I incubated my blot first with the first antibody of interest (let's call it "A") and after I developed the image, I incubated the blot O/N with the other antibody of interest ("B"). The thing is that both antibodies of interest come from the same host (rabbit), so, of course, my secondary antibody in both cases is anti-rabbit as well (the same). So, when I developed the image for the "B", I just saw the image of the first antibody ("A"). I understand this could be a possible outcome, although i had washed the blot after the "A" antibody really well.
Any ideas to avoid that problem?? (Stripping could help but unfortunately I cannot risk losing any protein) Do i just need to use different blots, one for each protein? :/

thanks!
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Western Blot Forum Immunola http://www.molecularstation.com/forum/western-blot-forum/87991-antibodies-interest-coming-form-same-host-troubleshooting.html
Salt effect on SDS page http://www.molecularstation.com/forum/western-blot-forum/87931-salt-effect-sds-page.html Fri, 14 Jun 2013 23:03:11 GMT I heard that salt concentration can affect the size a protein runs on SDS page. Is this true? And also, is it usually bigger or smaller or no way to...
I heard that salt concentration can affect the size a protein runs on SDS page. Is this true? And also, is it usually bigger or smaller or no way to tell?

This is a 4-12% Bis-Tris gel and its stained with coomassie.

Thanks
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Western Blot Forum scottishov http://www.molecularstation.com/forum/western-blot-forum/87931-salt-effect-sds-page.html