Questions pertaining to sodium dodecyl sulfate polyacrylamide gel electrophoresis, a technique used to separate proteins according to electrophoretic mobility

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Why my proteins are migrating like this on SDS-page?

I'm trying to make a SDS-page of total proteins extracted from algae but I'm encountering two problems. First, when I depose my proteins in the wells the sample goes down and then it starts to move up ...
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42 views

Why does migration distance depend on log of molecular weight in SDS-PAGE?

I really want to know why in the result of SDS PAGE, log of molecular weight(MW) and migration distance (distance from the loading well) have a linear relationship. Why is it log(MW) instead of MW? ...
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How is the acetone method different from QB buffer for extraction of plant protein?

I primarily work on animal tissue but I thought of running an SDS-PAGE for plant tissue just for fun. I searched for some protein extraction protocols online and I don't know which one to choose. One ...
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High intensity background on western blot membrane?

I did western blot and I got the high intensity background only in the upper part of the membrane and there is a horizontal line around 50-70kDa (this line separate the upper part and lower part of ...
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38 views

Does denaturing proteins lead to loss of epitopes?

I am doing an experiment where I have to do both Immunohistochemistry and SDS-PAGE. I am assuming that the native conformation of the protein is maintained in the IHC. But during the blot, we heat the ...
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154 views

Help analyzing SDS-Page gel

In this experiment, we transformed a truncation of the NFAT protein sequence into a plasmid vector to be expressed in E.Coli as a fusion protein with GST. We also attempted to transform the normal ...
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SDS-Page-Concentration confusion

So I purified some a protein, purified it using two different types of resins (comparing resins for cost efficiency). Ran a Bradford Assay to find concentrations of each. Unsure of SDS-Page protocol ...
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6k views

When running gels what is the difference between constant volts or constant amps?

In general, you want to be consistent with running your gels either at constant volts or constant amps. However, it is very clear that during the progression of both PAGE and agarose gels, the free ...
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Relative densitometry from SDS PAGE

I'd like to perform densitometry on a Coomassie stained SDS PAGE gel to compare a recombinant protein's expression levels under two conditions. I'm using BioRad's Image Lab software. My questions are ...
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65 views

On which amino-acids residue is the SDS acting on?

I would like to know exactly what is the mechanisme of the SDS, and I would like to know on which amino-acids residue the SDS is acting on. Can you help me please ? Thank you in advance !
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What is the purpose of using two layers of gel in SDS- PAGE?

I just made a SDS-PAGE with a top layer of stacking gel and a bottom layer of separating gel with different pH values of 0.5M Tris-HCl. The stacking was 6.8 and the separating gel was 8.8. What about ...
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What is SDS PAGE gel polymerization time?

I am working on 20% SDS PAGE. I want to know optimum polymerization time for 20% resolving gel and 6% stacking gel. If I increase the time then would it affect the band pattern?
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829 views

Troubleshooting SDS-PAGE of trypsin-treated BSA

I am currently working on SDS-PAGE technique having 20% acrylamide concentration for hydrolyzed BSA protein. Here I attached a gel photo for trypsin hydrolyzed BSA. I don't know , is it a good result ...
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351 views

Why not use SDS-PAGE as a method to detect viruses?

Recently, I have been researching about DNA and I know the most popular method for detecting viruses is based on DNA. After learning about proteins, I wonder why we do not detect viruses based on ...
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332 views

Does GTP-γS (GTP gamma S) bind all GTP-binding proteins?

I've just read an article Rab10 GTPase regulates ER dynamics and morphology - Nature Cell Biology 15, 169–178 (2013) doi:10.1038/ncb2647. In this paper, to identify Rab proteins in ER, first they ...
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1answer
525 views

Troubleshooting bioconjugates migration in a SDS-PAGE gel?

We do a lot of bioconjugation chemistry (click chemistry in particular but also NHS and Maleimide chemistries). Our method to valid the conjugation reactions have been to use SDS-PAGE gels followed ...
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639 views

Can I image Coomassie and GFP in gels at the same time with a fluorescence scanner?

I'm working with a GFP-tagged protein and am routinely using a fluorescence imager (GE Typhoon) and a standard optical scanner to capture fluorescent and absorption images, respectively, of my SDS-...
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1answer
10k views

How do SDS-PAGE gels differ in a Bis-Tris system vs. a Tris-Glycine system?

Protein migrate differently in Bis-Tris and Tris-Glycine gels. I was curious about the actual reasons why. Do certain gel systems result in a tighter resolution than others?
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What would cause proteins to get stuck in the stacking layer of a SDS-Page gel

Typically when proteins aggregate, they will get stuck at the top of the well. However, we're seeing some protein aggregate in the stacking layer even when we're treating the loading volume with DTT. ...
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6k views

How do Proteins migrate in MES vs. MOPS

My gels look significantly different in MES (2-(N-morpholino)ethanesulfonic acid) and MOPS (3-(N-morpholino)propanesulfonic acid). That is to be expected. What I don't understand is why the simple ...