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9

There is nothing like a general polymerization time, as this is a chemical reaction which depends on many factors. The most important are: APS: Concentration and and the age of the chemical are important. APS decomposes over time when it is not stored completely dry (which it is not on most benches) and also with freezing thawing cycles in storage. APS ...


6

Others have already explained the effect of different factors on polymerization time. This answer is mostly about: If I increase the time then would it affect the band pattern? It may. Some tips: Make a little extra mix so that you can leave some in the beaker to know if polymerization has happened. Always layer the poured resolving gel with water ...


5

This is the figure the question is about. On the right is the control experiment with GTP-γS, on the left without it: The bands that are visible in both experiments are unspecific binding. If GTP-γS doesn't affect their presence, the mechanism by which they bind to the column can't be specific to the GTPase functionality. The proteins the authors were ...


5

I'll answer only for SDS-Page, which is the system I am most familiar with. With a discontinuous buffer system, such as the well-known Laemmli system, resistance increases during electrophoresis, as (very mobile) chloride ions are replaced by glycinate (glycine ions). From Ohms law: Voltage (V) = Current (I) x Resistance (R) and the definition of ...


5

The stacking gel concentrates proteins loaded into the sample wells so that they are resolved as a unified "line" once they enter the stacking gel. The reason for the lower pH is that this "lower ionic strength implies higher electrical resistance and consequently a higher electric field, provoking the faster movement of the proteins and of every other ...


4

The other major difference between the two is the amount of acrylamide in the upper (stacking) gel - it's generally around 4%, while the lower (resolving) gel can vary from 6 or 8% to 20%, depending on the size of the protein(s) you're looking for. When you load your samples in the wells at the top of the gel, then start the current, not all of the sample ...


4

Put simply, the answer is that you could seek to detect viral proteins, but because these proteins would be very minor components of your sample, you would have to use an immunoblotting technique to detect a specific viral protein. While immunoblotting is quite a sensitive technique, I think its fair to say that it can be technically demanding. In contrast a ...


4

The polymerization time is strongly dependent on APS and TEMED concentrations. I do not think that the increased time for polymerization would somehow influence the division of the proteins in negative way. Actually the time varies from 20 min to 1 h, but I personally leave the gel to polymerize longer. Unpolymerized Acrylamide could build adducts with some ...


4

The first step in troubleshooting is to run controls - run lanes with your protein input alone, and the conjugate alone (you may need to play with the type/percentage of the gel if the conjugate molecule is much smaller than your target protein - attaching biotin to a 50 kDa protein, for example) to see if the smear shows up. Depending on the type of ...


3

At the interface of the stack gel and resolving gel is a pH change and a change in gel density. If you feel certain that your protein is not crosslinking so much that it can not enter the gel than I would think about the pH of the sample and the effect of Cu ions on the stacking effect of the stacingk gel. Is your sample the normal blue color (indication ...


3

I'm going to treat this as a partial homework question but provide some guidance as to how you can potentially address your question and have solid theory to back it up. Chymotrypsin preferentially cleaves peptide amide bonds where the carboxyl side of the amide bond (the P1 position) is a large hydrophobic amino acid (tyrosine, tryptophan, and ...


2

APS and TEMED concentrations matter a lot. Even with that standardized, your polyacrylamide on the shelf is losing reactive groups as we speak. Who know how long it sat in the warehouse? It all adds up to the fact that the minimum required time is practically impossible to predict. On the other hand, once polymerized, a gel is stable for days without ...


2

Well, to rationalize everyone's comments, I think @leonardo is right. This is a denaturing SDA PAGE gel. The migration of the SDS Micelles which are negatively charged, depends upon the shielding of the solution around it. The difference in mobility is because the SDS micelles will experience a slightly different field at pH ~6.2 (MES) vs 7.2 (MOPS). ...


2

SDS PAGE system rely on the fact that protein is denatured and surrounded by the SDS negatively charged detergent micelle. This eliminates most of the charge and idiosyncratic solubility differences from one protein to another and gives a reasonable separation based only on size of the protein which is related to the size of the SDS micelle around each ...


1

Sounds like the copper cross linking the protein or creating aggregates that the SDS buffer can't break up. add EDTA to your loading buffer before you cook it?


1

Coomassie blue fluoresces in infrared when bound to protein, so if your reader has the appropriate filter set, it should be possible.



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