Hi Rockstar. I am not really sure what you mean by inactivate the bead. If you want treat the beads so that they wont specifically bind to His-tagged protein to look at nonspecific binding to the agarose, then just remove the Ni2+. Use EDTA to do this.
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Hi Mariam. The sulfhydryl groups on your cysteines will react and cross-link with each other as long as they are in an oxidative environment. Predicting which 2 of the 3 will form the intrachain disulfide bond is not possible with the information you have provided, but if you were to express this protein you may end up with more than one combination of disulfides, e.g. Cys1 linked to Cys3 in one molecule and Cys2 linked to Cys3 in another. Unless one of the sulfhydryl groups is completely inaccessible, you can not really predict which disulfides will form.
Hi Veevaa. Sorry, just noticed your question. The design of your experiment seems to require that you know if both the protein binding site and ligand (compound) binding site are present in your expression constructs. In this case, searching the literature and comparing the sequence of other members of the protein family you are studying may provide you with some confidence that you have included both binding sites. Another option is to make multiple constructs and test them out.
Hope this helps.
Hi pkay. Further characterization of your refolded protein may help you rule out non-specific binding to the cells. After refolding how do you analyze the activity of the protein? You mentioned there is a ligand that binds, but do you have titration curves and affinity calcuations of this binding to compare to the native non-truncated membrane protein? Also, reduced vs. non-reduced samples by SDS-PAGE, running a native gel or analytical scale SEC can provide data about possible multimerization and aggregration of the refolded proein. If the refolded protein isn't in its normal state (eg monomer or homodimer), then the binding you see may not be real.
I have always had issues with size exclusion columns probability due to the battle between diffusion and resolution. Is there are reason you are using size exclusion as a second chromatographic step rather than ion exchange?
Hi Missle. I would recommend taking time points (add SDS without extra reducing agent and freeze) during storage, and then running "non-reduced" samples on a gel. I believe disulfide exchange will occur spontaneously as the protein molecules interact and if so, you can see evidence of multimer formation on the gel.
Hi Veevaa. A single domain from a multi-domain protein can be expressed and used to study protein-protein interactions. As long as you are expressing the domains that interact with each other, the interaction can still occur.
Hi Trang. Unless your plasmid was also pUC19 and you used the exact same amount, then you are not really looking at a comparable control. Colony counts can be interesting to do, but they rarely provide any useful information. The results from a screen (either digest or PCR) of colonies on your experimental plate are more important.
Having protein that is too concentrated is a great problem to have. You can load 1uL on a gel if you have the right kind of syringe. Otherwise, I would dilute the sample in 1X sample buffer just prior to loading the gel - no need to add extra salts, buffers, etc. that are likely in the extraction buffer to the sample.
Hi Missle. The decision to "cap" the protein will likely depend on your expression system, method of purification, and the activity of cleaved vs. uncleaved protein. You could always start expressing the wild-type protein and determine along the way if protease activity is an issue.
Here is a paper I found about a protease that cleaves after a penultimate proline residue:http://www.pnas.org/...7/3472.full.pdf
This might be what you were thinking of.
Hi Stive. I haven't encountered this exact problem, but I have attempted other affinity chromatography columns that didn't work. Are you able to detect the 20kD-biotin protein in the lysate and the flow through in a manner that would indicate is actually is biotinylated? Is your control protein expressed in the same BL21-BirA cells?
The length of the linker would depend on the downstream experiments that are to be done with the protein. For example, if the protein is going to be immoblized for a binding assay, i would use a longer linker (8-10 amino acids) to attempt to create more space between the protein molecules to allow binding partners or ligands access to binding sites. If the tag is simply for purification purposes or used as an epitope, then a linker may not be necessary.
I have had many isssues with anti-FLAG Western blots (not sure if that's what you are using to detect expression) and always run a control for the primary antibody on each blot.
I have seen rounded or dot-like bands before using EtBr when there is a lot of DNA present in the band. The lanes on the gel appear to be overloaded as the smearing above and below your bands of interest indicate a loss of resolution. Have you tried to extract the fragments and then run a diluted sample or smaller load on a gel?
I have never compared precipitation methods to be able to comment on the advantages of one over the other. I would routinely use TCA precipitation with a final acetone wash step to dry the sample, but these samples were simply used for SDS-PAGE and nothing else. After lysis of of your cells, DNaseI should be added to the lysate. After sufficient time for enzyme to work, the viscosity due to genomic DNA is no longer an issue. I would hesitate to remove anything with a pipette tip as a significant percentage of your protein may be present in what you are removing.
Hi guyava. In my experience, using a non-native secretion signal should not be an issue as long as the cell line recognizes it. Not including the propeptide sequence should not affect dimerization either since dimerization will occur between the two mature 12.5 kDa molecules in TGFb – nothing to do with the propeptide. The activity will have to be determined experimentally.
In general, predictive calculations can be helpful when figuring out the best strategy to express a protein, but there is still no substitute for experimental data. So does it matter? Not really. What's more important in your case is probably, 1) where you are targeting the expression of this membrane protein and 2) the manner in which you determine protein expression levels.