Hi, every one,

Now, I am working on a protein, in the N terminal, there is one Cys, in the other flank, another Cys residue exists. I plan to use BM(PEO)3 to conjugate themonomer to polymers via disulfide crosslinkg.

First, I dilute the BM(PEO)3 into DMSO and the final concentration is 10mM/L. At room temperature, I add the BM(PEO)3 to my protein(4mg/ml) stock, the final molar ration of BM(PEO)3 : my protein is 1:1. Then I leave the protein solution in room temperature for 1 hour even longer time. In most of the time, I only get dimer, and could not get octamer.

How can I get a longer polymer? Any tips are appreciated
Best regards
biomd

how can we isolate protein from cells which are growing on collagin matrix?because as collage itself a protein.
Kindly reply me as soon as possible.
Thanks

In article <mailman.32.1254676405.1133.proteins@net.bio.net >, elham
rastegari <elham.rastegar@gmail.com> wrote:


I am sure that what is causing your problem (darkening to black) is
residues of polyphenolics, biphenolics, etc. Most land plants have enzymes
called polyphenol/biphenol oxidases which cause huge problems when
attempting to purify RNA or DNA (and apparently protein). I have had a
moderate amount of experience in isolating (or trying to isolate) usable
nucleic acids from ferns, mosses, a liverwort as well as gymnosperms and
angiosperms. Generally, flowering plants that are food sources tend to be
low in polyphenols so they are usually easier to work with. Curculigo
latifolia (Molineria latifolia) is known for its protein curculin (a
potential artificial sweetener) but I would suspect the fruit does contain
lots of polysaccharides as well as apparently the phenolic compounds.
I think you have a challenging problem here. I know how nucleic acid
extraction could probably be accomplished but what you want (the protein)
is normally in the part you throw out in nucleic acid extractions, and
phenolic compounds are also normally in the organic phase except for those
permanently cross-linked to the RNA or DNA that you may be trying to
purify.
Certainly keeping a reducing agent like mercaptoethanol or DTT in the
solutions will inhibit the phenol oxidases, but I think the whole protocol
will have to be rethought to attempt to separate the protein fraction from
the phenolics as early as possible.
Your difficulties are certainly not unexpected or unique because they are
faced frequently by anyone trying to do much molecular biology on plants.

Cheers,

David

Dear all,

I have a small peptide (~20a.a) without any tryptophan and tyrosine, so I cannot measure its absorbance at 280nm to determine the concentration. Apart from Bradford assay, is there any good method to determine my peptide concentration?

Thanks for your help

Hi,

I recently started extracting whole proteins from human adipose tissue but the problem is that I am not able to get rid of blood in the samples even after washing in PBS and the lysate shows red colour. Guess this might play a role in protein estimation as the color plays a major role in it. I'm using BCA kit for that. Eager for any suggestions.

Thanks.