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dimensionsbio's Content

There have been 16 items by dimensionsbio (Search limited from 20-February 19)

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#167126 how do you inactivate His-tag affinity beads??

Posted by dimensionsbio on 18 April 2014 - 03:30 AM in Protein Expression and Purification

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.

#165703 Modeling of a protein with disulfide bond

Posted by dimensionsbio on 03 March 2014 - 08:09 PM in Bioinformatics and Biostatistics

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.

#165466 Just part of the CDS (like transmembrane domain) or the full-length CDS for prot

Posted by dimensionsbio on 25 February 2014 - 04:30 AM in Protein Expression and Purification

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.

#165390 purified protein binds cell surface. how to be sure it is not an artifact?

Posted by dimensionsbio on 23 February 2014 - 04:18 AM in Protein Expression and Purification

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.

#164627 Sephacryl S-300 didn't enhance the purity of my enzyme

Posted by dimensionsbio on 29 January 2014 - 07:33 PM in Protein Expression and Purification

Hi Biogareth,


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?

#164260 Will 5mM DTT break existing disulfide bonds?

Posted by dimensionsbio on 16 January 2014 - 06:42 PM in Protein Expression and Purification

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. 

#164147 Just part of the CDS (like transmembrane domain) or the full-length CDS for prot

Posted by dimensionsbio on 14 January 2014 - 03:56 PM in Protein Expression and Purification

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.

#164049 bacterial transformation kit life-technologies

Posted by dimensionsbio on 11 January 2014 - 08:35 AM in Molecular Biology

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.

#162806 cytoplasmic protein

Posted by dimensionsbio on 25 November 2013 - 10:36 AM in Molecular Biology

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.

#162659 Does a C-terminal Proline Residue Increase Degradation Risk?

Posted by dimensionsbio on 21 November 2013 - 02:37 AM in Protein Expression and Purification

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.

#162101 Problems with biotynylated protein binding to monomeric avidin

Posted by dimensionsbio on 04 November 2013 - 02:46 PM in Protein Expression and Purification

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?

#161419 designing fusion protein with Flag tag

Posted by dimensionsbio on 18 October 2013 - 12:19 PM in Protein Expression and Purification

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.

#161169 Dotted bands on my DNA agarose electrophoresis gel.

Posted by dimensionsbio on 12 October 2013 - 04:20 AM in Genetics and Genomics

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?

#160927 Protein precipitation method

Posted by dimensionsbio on 04 October 2013 - 12:37 PM in Protein and Proteomics

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.

#160741 Recombinant expression of cytokines in mammalian cells without the propeptide re

Posted by dimensionsbio on 28 September 2013 - 01:16 PM in Biochemistry

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.

#160723 instability index of expressed protein

Posted by dimensionsbio on 27 September 2013 - 10:45 AM in Protein Expression and Purification

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.

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