5
votes
Accepted
How does SDS-PAGE separate based on mass?
You are correct that molecule mobility depends on the mass-to-charge ratio, and this means that different sized molecules with the same $\frac{m}{q}$ will have the same acceleration. However, the ...
3
votes
Accepted
Electrodes in non-conducting fluid? Are they neutral? Electrolysis, Electrophoresis
Electrodes are neutral, but they are held at different potential. This is similar to the current flow in a wire: although current flows, the wire remains neutral, i.e., no charge is accumulated (...
3
votes
Accepted
What is meant by 4 –12% or 8% SDS-PAGE?
Do the percentage values refer to the percentage of acrylamide in the gel?
Yes. The 8% gel is 8 g acrylamide per 100 mL. The “4-12%” gel is a gradient gel, which are useful for separating proteins ...
3
votes
Accepted
In SDS-PAGE, why are the sample buffer and running buffer different in concentration?
The reason why the sample buffer is more concentrated (typically 2x or 5x depending on your protein concentration) is its dilution when you mix it with the sample. You mix 4 parts of your sample and 1 ...
3
votes
What kind of "size" does SDS-PAGE separate by?
The protocol for SDS-PAGE uses a solution of sodium dodecyl sulfate (SDS, also known as lauryl sulfate) to solubilize and linearize folded protein molecules and give them a negative charge that is ...
3
votes
Protein bands not visible after sds page coomassie dye stain
The problem here is concentration. Coomassie Blue stain can only detect protein band greater than or near to 50ng, in this case, the concentration of your protein is too low for detection. If you want ...
2
votes
Why my proteins are migrating like this on SDS-page?
It's a guess, as I haven't run a algae sample on SDS-PAGE, but your migration problems might be due the algal polysaccharides that are present in your sample. Check if your protein purification ...
2
votes
Why does migration distance depend on log of molecular weight in SDS-PAGE?
I think this is just an empirical formula that (approximately) fulfills the non-linear response of the relative migration distance (Mr) with the molecular weight (MW) - an observational rather than ...
2
votes
Why does migration distance depend on log of molecular weight in SDS-PAGE?
An SDS-PAGE standard curve of Rf vs LogMW is linear because the standard you're given is engineered to behave that way. An expanded SDS-PAGE curve would show that the actual graph is sigmoidal: ...
2
votes
Can denatured GFP show fluorescence?
According to PDB (protein data bank — a repository for protein structures) the chromophore must be protected from interaction with water molecules to fluoresce.
The chromophore is found right in ...
2
votes
Preparing sample for SDS PAGE
Usually you would want to keep the amount same, not the concentration. However, if you still want the concentration to be the same then you can add suitable amounts of PBS or your lysis buffer.
For ...
1
vote
Mesh formation in the wells of SDS PAGE. How to solve it?
This issue is driving me crazy too. Although I haven't identified the exact cause, here are the steps I've taken to minimize mesh formation:
(1) Prepare your gel on the same day you plan to perform ...
1
vote
gel band contrast
If this is accepted or not depends on your institution. For publications, it is usually accepted to use adjustions of color, tonal values and contrast which are applied to the whole image and which do ...
1
vote
Blackgram leaves. SDS page. Phosphate buffer method
You should de-stain your gel longer. I can see at least one thick band at the top, but without de-staining longer you are probably missing other fainter bands.
Also, please explain what you mean by "...
1
vote
Does heating proteins before a SDS-PAGE gel effect gel result?
As far as I know, it has an effect. If you denature your protein before running the gel, you do a normal SDS-PAGE and seperate the proteins by their size. Since you use SDS, the charge of the protein ...
1
vote
Accepted
Does denaturing proteins lead to loss of epitopes?
Short Answer - If the same antibody works both for IHC and SDS-PAGE it either means it is monoclonal against a surface linear epitope or a polyclonal antibody.
Long Answer -
Starting off, there are ...
1
vote
Accepted
Help analyzing SDS-Page gel
Here are some thoughts based on my own experiences purifying many proteins and running many SDS-PAGE gels:
(1) Resolution--how fast did you run this gel? Often if you run at higher than say 150V or ...
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