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6

Western Blot tests on young children are practically useless, since they test for antibodies. The child will likely have antibodies passed down by the HIV+ mother, regardless of whether the child has HIV. The test will show the antibodies, which may be mistaken for an active immune response from the child. As such, there will be a high false-positive rate ...


5

Blocking buffer Once the proteins in the gel have been transferred to the nitrocellulose membrane it is necessary to coat the rest of the surface of the membrane with an unrelated protein. This is necessary because all proteins will bind non-specifically to the nitrocellulose. Once the membrane has been blocked the only way that antibiody proteins (the ...


4

Doing westerns with primary tissue can be tough, especially because of the presence (sometimes at high levels) of extracellular matrix (ECM). This material can be quite resistant to homogenization and some lysis buffers. One method I have found to work is to snap-freeze the tissue in liquid nitrogen (not on dry ice or at -80°C) directly after removal ...


3

I do not think that a publication quality blot should have such an artifact, but I was able to find something similar by purposely over blotting (not the same as over exposing) a gel. If you use too much primary, secondary, or developing reagent, you can get your HRP signal to "burn in" a membrane where you get a distinct "negative band." By negative ...


2

Vinculin! I love our grad-students, I can't believe I didn't think of it last night. Also, awesome chart, even if it's from a company.


1

So we went ahead and did this. The efficiency was much lower, which you might expect, and dehydration did aid the process. We found that taking raw lysates and running them through the linked protocol, had about ~20% the efficiency of conducting it on purified protein. I assume the majority of the loss was due to reagents being expended on proteins that ...


1

Here's an example of a chemiluminescent blot: For those who care, the first column on the left is the weight standard, the next two are pooled pellets, and the rest are increasing fractions of purified HIV using a mouse monoclonal anti-p24 (capsid) antibody. This blot is pretty clean (although for my work it was pretty disappointing...) but here's one ...


1

So after a lot of work in optimization, I thought I would post what worked best for me. This buffer recipe was able to successfully transfer EGFR and insulin from the same lysate, and a clear band for both (large and small protein respectively). 10% SDS-PAGE gels were transferred at 1A for 10 min. High Current Transfer Buffer 48 mM Tris 15 mM HEPPS 1.0 ...


1

The one that actually makes me skeptical of the use of this is that this "cure" was primarily a massive dose of antiretrovirals used as catchup therapy because the infant's mother didn't have access to prophylactic treatments while she was pregnant and delivering. Basically, it's a cure that requires you to have access to antivirals very, very near the ...



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