protein size determination? - (Feb/26/2008 )
QUOTE (mdfenko @ Mar 13 2008, 11:11 AM)
QUOTE (DNA Gyrase @ Mar 13 2008, 06:32 AM)
hello, I haven't read full text of the article but I seriously doubt it worth it because they not only didn't refer to it in abstract but as you said they didn't refer to it in their results and only they refer to it in discussion(a sort of speculation I presume).
Now back to the facts, charge of proteins is the single most important factor in mobility in PAGE. yes, size, shape is also important but charge is driving force and is most important factor( I haven't invented these, you can find it in all biochemistry text books e.g. stryer). you may have proteins that have negative net charge & proteins that positive net charge. If you change pH from neutral, then you may denature some proteins. If OP dose not know anything about this protein and how these proteins may have attached to form a dimer then changing pH can be as much damaging as using SDS because possible quaternary structure may are held by ionic forces. I don't know what they have written in discussion section of that article but I presume it was an specific case and they had some information about their protein and with the help of PAGE on combining its results with other results they could reach an ESTIMATE of size. All I want to say it is never can be generalized for unknown proteins because there are solid rock reasons that is mentioned in every text book that can't be challenged in discussion section of a faint article from 1991.
cheers,
DG
Now back to the facts, charge of proteins is the single most important factor in mobility in PAGE. yes, size, shape is also important but charge is driving force and is most important factor( I haven't invented these, you can find it in all biochemistry text books e.g. stryer). you may have proteins that have negative net charge & proteins that positive net charge. If you change pH from neutral, then you may denature some proteins. If OP dose not know anything about this protein and how these proteins may have attached to form a dimer then changing pH can be as much damaging as using SDS because possible quaternary structure may are held by ionic forces. I don't know what they have written in discussion section of that article but I presume it was an specific case and they had some information about their protein and with the help of PAGE on combining its results with other results they could reach an ESTIMATE of size. All I want to say it is never can be generalized for unknown proteins because there are solid rock reasons that is mentioned in every text book that can't be challenged in discussion section of a faint article from 1991.
cheers,
DG
as you say, you haven't read the full text of the paper so you don't know what it said about size separation, you just assume that it is inconsequential. i referred you to the discussion because it gave a more succinct explanation. size separation is written about in the introduction and results, as well.
a better reference is the chapter from "methods in cell biology" from 2001. shagger explains how size separation is selected for and is, in fact, the overriding factor with bn-page (last, long, paragraph in the introduction). in the section about applications he states (and shows with a picture of a gel) that bn-page can be used to determine the masses of native protein complexes (as i pointed out before, a second dimension is added to show the subunit composition of the complexes separated with bn-page).
native page has been in use for a long time. a lot is known about the mechanisms of separation involved. while it is true that charge is the major factor for separation, it is also true that other factors can be selectively enhanced by various methods. one selectable factor is size. this can be selected for by adjusting the porosity of the matrix. in this way, two proteins of equal charge but different size can be separated. this is why crosslinked polyacrylamide was selected as the major matrix for protein electrophoresis.
nucleic acids all carry the same charge density. if you subject them to an electric field without a matrix then all of the molecules, regardless of size, will migrate together. put them into a porous matrix (page or age) and you will separate the molecules by size.
be careful when you read the texts or you'll end up like my children who read part of something or hear part of something from their teacher and then argue that there is no more to the issue.
Hello,
I don't have prompt access to the article but I think in ocean of articles, an article should really worth it to spend time for finding it and reading it. My argument was if that article has introduced something so revolutionary that all biochemistry textbooks that I have read(stryer, rawn, harper, voet) and many other books say something in contradiction of it why after 15 years in still has not entered biochem text books? And why we should argue about what that article said and the other article said something else. Why you don't present reasons why and how charge of a protein can be neglected in PAGE rather than referring me to full text of an article that I don't have prompt access to it?
If the protein in question happens to have 2 times or 4 times or x times more charge than your marker how you want to draw useful size information from it?
Give me just one protocol of size determination using PAGE that can be applied to any unknown protein. Hopefully this time you don't refer to me to an article from 1970 that I don't have prompt access to full text of it. Or at least present reasons and enlighten me how are you going to draw useful size information from an unknown protein preserving its native conformation using PAGE.
cheers,
DG
-DNA Gyrase-
QUOTE (DNA Gyrase @ Mar 13 2008, 05:39 PM)
Hello,
I don't have prompt access to the article but I think in ocean of articles, an article should really worth it to spend time for finding it and reading it. My argument was if that article has introduced something so revolutionary that all biochemistry textbooks that I have read(stryer, rawn, harper, voet) and many other books say something in contradiction of it why after 15 years in still has not entered biochem text books? And why we should argue about what that article said and the other article said something else. Why you don't present reasons why and how charge of a protein can be neglected in PAGE rather than referring me to full text of an article that I don't have prompt access to it?
If the protein in question happens to have 2 times or 4 times or x times more charge than your marker how you want to draw useful size information from it?
Give me just one protocol of size determination using PAGE that can be applied to any unknown protein. Hopefully this time you don't refer to me to an article from 1970 that I don't have prompt access to full text of it. Or at least present reasons and enlighten me how are you going to draw useful size information from an unknown protein preserving its native conformation using PAGE.
cheers,
DG
I don't have prompt access to the article but I think in ocean of articles, an article should really worth it to spend time for finding it and reading it. My argument was if that article has introduced something so revolutionary that all biochemistry textbooks that I have read(stryer, rawn, harper, voet) and many other books say something in contradiction of it why after 15 years in still has not entered biochem text books? And why we should argue about what that article said and the other article said something else. Why you don't present reasons why and how charge of a protein can be neglected in PAGE rather than referring me to full text of an article that I don't have prompt access to it?
If the protein in question happens to have 2 times or 4 times or x times more charge than your marker how you want to draw useful size information from it?
Give me just one protocol of size determination using PAGE that can be applied to any unknown protein. Hopefully this time you don't refer to me to an article from 1970 that I don't have prompt access to full text of it. Or at least present reasons and enlighten me how are you going to draw useful size information from an unknown protein preserving its native conformation using PAGE.
cheers,
DG
you don't want to obtain the article but you'll refute it blindly.
i first pointed to an article from 1991 then a chapter from 2001 but you argue that it is from 1970 and beneath you to look at.
okay. since you don't want to do the leg work (it took me one day to get them from our library and inter-library loan, and only that long because our librarian is semi-retired and not always available) then i'll fill you in.
shagger says that the coomassie brilliant blue g-250 that is used in bn-page binds to and imparts a negative charge to the protein, similar to sds but without denaturing the protein or complex (the proteins retain biochemical activity). he further enhances size separation by using a gradient gel. he says that standard proteins fit a general calibration curve (from an earlier reference).
text books are periodically revised as new and corrected information becomes available. they give theory but may leave out exceptions so as not to confuse students. i fear that it has, anyway.
-mdfenko-
Thanks for your comments. Could you tell me the exact reference for the "methods in cell biology" 2001 that contains the chapter written by Shagger. I found it interesting and would also like obtain it from our library.
I have one more question: You mentioned 2-mercaptoethanol and DTT as agent that can disturb the structure of native proteins. I am now confused since I have used both of these agents in my extraction buffers and buffer in enzyme activity tests since they are used by many authors in their protocols. I have read that DTT can enhance the activity of proteins and even restore the activity of proteins in the presence of inhibitors.
I also read that many proteins require free sulfhydryl groups to maintain their biological activity and need to purify in the presence of reducing agents such as mercaptoethanol. Is this true or is it just a matter of how much is used?
-sarita-
QUOTE (sarita @ Mar 22 2008, 04:48 AM)
Thanks for your comments. Could you tell me the exact reference for the "methods in cell biology" 2001 that contains the chapter written by Shagger. I found it interesting and would also like obtain it from our library.
I have one more question: You mentioned 2-mercaptoethanol and DTT as agent that can disturb the structure of native proteins. I am now confused since I have used both of these agents in my extraction buffers and buffer in enzyme activity tests since they are used by many authors in their protocols. I have read that DTT can enhance the activity of proteins and even restore the activity of proteins in the presence of inhibitors.
I also read that many proteins require free sulfhydryl groups to maintain their biological activity and need to purify in the presence of reducing agents such as mercaptoethanol. Is this true or is it just a matter of how much is used?
I have one more question: You mentioned 2-mercaptoethanol and DTT as agent that can disturb the structure of native proteins. I am now confused since I have used both of these agents in my extraction buffers and buffer in enzyme activity tests since they are used by many authors in their protocols. I have read that DTT can enhance the activity of proteins and even restore the activity of proteins in the presence of inhibitors.
I also read that many proteins require free sulfhydryl groups to maintain their biological activity and need to purify in the presence of reducing agents such as mercaptoethanol. Is this true or is it just a matter of how much is used?
the reference is:
Shagger, H. (2001). Blue-Native Gels to Isolate Protein Complexes from Mitochondria. Meth. Cell Biol. 65: 231-244.
as for 2-me and dtt:
in high concentration they will break disulfide bonds, at low concentration they will maintain sh groups. we use 0.5-2mM dtt to maintain most of our proteins.
-mdfenko-