Questions tagged [sds-page]

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

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Mesh formation in the wells of SDS PAGE. How to solve it?

I casted 10% resolving gel and 4% stacking gel and every time a mesh like structure ( polymerisation) in the wells are formed.How to avoid this from happening and how do i get clear wells? If someone ...
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Electrodes in non-conducting fluid? Are they neutral? Electrolysis, Electrophoresis

I'm a moleculary biology student and have to do a detailed presentation about SDS-PAGE for my next lab. So I was currently trying to understand the physics of electrophoresis and electrolysis. Since I ...
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What is meant by 4 –12% or 8% SDS-PAGE?

I am reading a journal paper and I am looking at the materials and methods section. Regarding the Western blot method in the paper, I have come across the following statement: Proteins were separated ...
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Why is my DNA band bulging?

This is the only image my the TA was able to get for us. And, we're using it for our lab report. The image isn't even ours. It's another group's image that we're sharing. But I don't understand why in ...
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How does SDS-PAGE separate based on mass?

In SDS-PAGE, electric field and mass-to-charge ratio are approximated to be constant for all proteins. Also, if $F=qE =ma$, then $\frac{m}{q}a=E$. Thus, all proteins must migrate with a constant ...
Eliot Behr's user avatar
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Can denatured GFP show fluorescence?

GFP ( green fluorescent protein) can show green fluorescence. And its fluorescence is due to the tri peptide chromophore which is given in below I was wondering, can we observe fluorescence, if we ...
Science123's user avatar
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Preparing sample for SDS PAGE

I have more than 10 cell lysate samples (70 µL each) whose concentration varies from 1.9 mg/mL to 4.8 mg/mL. I have 5X and 2X SDS sample buffers. I would like to prepare SDS PAGE samples in such a way ...
Science123's user avatar
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How can I differentiate between polysaccharide bands and protein bands on SDS-PAGE? [closed]

I tried to extract bacterial polysaccharide but after running a SDS-PAGE I couldn't differentiate between the polysaccharide band and protein ones. I face a problem of moving up of the samples from ...
S.masoud's user avatar
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gel band contrast

Is there any way to increase the contrast of my SDS PAGE gel. I want to increase the coomassie stained gel contrast of my gel bands a little bit as it looks little less for my thesis. Ive heard that ...
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Protein bands not visible after sds page coomassie dye stain

I am working with cell wall protein of Candida albicans and cryptococcus neoformans but after protein extraction and run on SDS PAGE my bands are not visible after coomassie staining. Can anyone tell ...
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Blackgram leaves. SDS page. Phosphate buffer method

I am doing protein analysis in blackgram leaves by using phosphate buffer method. But I cannot get proper bands on my SDS-PAGE. What should I do to get proper bands?
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Laemmli-SDS-PAGE problems [closed]

I did Laemmli-SDS-PAGE for my Ammonium sulphate precipitate but I had very weak band and have very weird part at the end of gel. Please help me to solve that problem. Thanks
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In SDS-PAGE, why are the sample buffer and running buffer different in concentration?

I'm currently trying to understand the concepts of SDS-PAGE. Every protocol I've read says to use 5X for sample buffer and 1X for running buffer, but I don't really understand for what reason? What ...
Migs V.'s user avatar
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What is/are the effect(s) of altering the Tris concentration on polyacrylamide gels?

Why do we use two different concentrations of Tris (i.e. 1.5M Tris in resolving gel and 1.0M Tris in stacking gel)? For the gel to run properly there needs to be a different pH, but that can be done ...
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What kind of "size" does SDS-PAGE separate by?

What is the exact property of the protein that affects migration of bands in SDS-PAGE; is it length, volume, or mass? For example, will GGGGGGGGGG (glycine 10-mer; ...
oryza's user avatar
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Does heating proteins before a SDS-PAGE gel effect gel result?

I need to isolate a protein that a bacteria has excreted into an agar plate. My plan is cut out parts of the agar, heat the agar to melt it and then concentrate the protein through some purification ...
A. Radek Martinez's user avatar
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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 ...
Radioactivestar88's user avatar
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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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7 votes
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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? ...
Eiswein.Y's user avatar
1 vote
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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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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 ...
PrinceAladdin28's user avatar
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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 ...
bravetang8's user avatar
1 vote
1 answer
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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?
kt123's user avatar
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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 ...
kt123's user avatar
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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 ...
DucFabulous's user avatar
6 votes
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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 ...
Armacino's user avatar
7 votes
2 answers
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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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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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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 ...
bobthejoe's user avatar
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3 votes
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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 ...
bobthejoe's user avatar
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7 votes
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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 ...
bobthejoe's user avatar
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6 votes
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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?
bobthejoe's user avatar
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