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7

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 ...


6

ImageJ doesn't have a feature to remove individual lanes. But that shouldn't be a problem. All you have to do is draw the first lane correctly (I'm referring to size). Then press 1. Now, while the selection is still... selected, click inside it, but not on the number (where the cursor becomes hand), and drag it where you want the next lane. And press 2. And ...


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

What you can do depends on your proteins: If both proteins (your target protein and the loading control) are seperated far enough, you can detect both of them in the same step by adding both primary antibodies and both secondary ABs into the buffer at the same time. They will bind only to their specific epitope and you will get nice signals. This is ...


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

It is a common practice to prove a result using an orthogonal technique. Like RNAseq followed by qRT-PCR etc. Western blotting is not a robust technique and cross comparisons are difficult because of difference in the avidities/affinities of different antibodies. So comparisons can be made only with one protein-control pair in different conditions. LC-MS ...


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.


2

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 ...


2

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 ...


2

I think that enzyme-linked immunosorbent assay (or ELISA) is the best way to do so, given you have an antibody against the protein to coat the sample wells with... You could use mice to create polyclonal antibodies against your protein by injecting the protein and collecting the serum and purifying it...as long as there isn't similar proteins to the ...


2

Sample preparation for protein gels is not a complex task. Simply mix the appropriate amount of sample buffer with your sample and load it. For a 2x sample buffer use equal amounts of sample and buffer, for 5x sample buffer use 4 parts of sample and 1 part of buffer (for examle 40µl + 10µl). Heat the mixed samples for 5 minutes at 95°C, cool them immediately ...


2

In my opinion you should use this formula: $\huge \frac{log_2(Iso_1/Iso_2)}{log_2(GAPDH)} $ This will normalize the relative fold differences between the isoforms with the loading control- GAPDH. Since both numerator and denominator are log transformed they are in comparable domains unlike the formula-2 that you mention in your question. This is just a ...


2

There is no evidence that one is better than the other, most likely because it differs from case to case. Neither you, nor your critics, are right. There is a tiny bit of science in a paper on digitizing blots, generalizing from blots of a specific protein (PMID: 19517440), and they use grayscale for no given reason. Come to think, that is the best paper in ...


1

In general, leupeptin (a cysteine, threonin and serine protease) and aprotinin (sometimes called Trasylol; trypsin inhibitor) are included in most mammalian cell lysate buffers. Whether you need them is really up to your protein of interest: if you know how it gets degraded in vitro, then you should be fine with inhibition of just those proteases. If instead ...


1

Try rinsing the blot in TBS or PBS before treating. Shake it try and place on level dry surface. When I pour the a chemo reagent on there I just pour enough so the surface tension keeps the fluid on the paper not allowing it to run off. Other possibilities I see could be in your transfer. The temp could not be uniform in the buffer


1

Well usually in an ECL kit, one of the reagents is the substrate and the other one is hydrogen peroxide, which activates the substrate for breakdown using the HRP on your secondary antibody (Ab) so I genuinely doubt what you are seeing is the result of ratio difference. In order to make that conclusion you need to run your samples twice (on two separate ...


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 ...



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