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Hi there
I'm looking at doing a sandwich culture to mimic the hypoxic conditions of a tumor. I wanted to know if anyone has any suggestions for how I can stain the cells for HIF-1a after they have been fixed?
Thanks
Alex
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Uneven beta actin
15 April 2013 - 05:52 PM
Hi there
I haven't been able to get even beta actin bands. I have had other, much more experienced people in my lab who have no issues like this load my samples and they still get uneven bands too. This leads me to think that it's something other than my loading which is causing the issues. I don't think that it's my samples, because the band intensity of one sample might be low on one gel, but the same sample on another gel might be really intense. Does anyone have any suggestions? I've included two images, they are the same samples run on two different gels so you can see how they look.
Picture1.jpg 3.87K
34 downloads The gels are 8%, I run at 80v for about 2 hours and I transfer them overnight at 30v onto pvdf membranes. I block with 5% skim milk, incubate with primary at 1:10000 for 2 hours, wash, do secondary for 2 hours and then visualise using our chemidoc system.
Thanks!
I haven't been able to get even beta actin bands. I have had other, much more experienced people in my lab who have no issues like this load my samples and they still get uneven bands too. This leads me to think that it's something other than my loading which is causing the issues. I don't think that it's my samples, because the band intensity of one sample might be low on one gel, but the same sample on another gel might be really intense. Does anyone have any suggestions? I've included two images, they are the same samples run on two different gels so you can see how they look.
Picture1.jpg 3.87K
34 downloads The gels are 8%, I run at 80v for about 2 hours and I transfer them overnight at 30v onto pvdf membranes. I block with 5% skim milk, incubate with primary at 1:10000 for 2 hours, wash, do secondary for 2 hours and then visualise using our chemidoc system.Thanks!
Making an acetate buffer
26 February 2013 - 04:15 PM
Hi
I was wondering if anyone can explain to me how to make buffers? I have very limited chemistry knowledge, so the henderson hasselbach is a bit over my head.
Specifically, I want to make a 150 mM acetate buffer at pH 5.0.
Thanks
I was wondering if anyone can explain to me how to make buffers? I have very limited chemistry knowledge, so the henderson hasselbach is a bit over my head.
Specifically, I want to make a 150 mM acetate buffer at pH 5.0.
Thanks
Some bands not coming up, others uneven
21 February 2013 - 03:50 PM
Hi there
I have an issue with my blots. I had been getting good results using these lysates, but now I am not getting any bands coming up for the proteins I am probing for, and only some bands coming up for beta actin (some are faint, some are normal). I have attached a picture of the most recent blot. People in my lab have suggested that the membrane may not be getting soaked in the antibody properly. Does anyone have any suggestions, or has seen something like this before?
Thanks
2013.02.21 low res.pdf 17.08K
80 downloads
I have an issue with my blots. I had been getting good results using these lysates, but now I am not getting any bands coming up for the proteins I am probing for, and only some bands coming up for beta actin (some are faint, some are normal). I have attached a picture of the most recent blot. People in my lab have suggested that the membrane may not be getting soaked in the antibody properly. Does anyone have any suggestions, or has seen something like this before?
Thanks
2013.02.21 low res.pdf 17.08K
80 downloads
