protein size determination? - (Feb/26/2008 )
QUOTE (sarita @ Mar 2 2008, 10:24 PM)
The purpose is to see if the protein is dimer. So I cannot use SDS-PAGE because it will denaturate the protein. Unfortunately I dont have access to gel filtration HPLC. So I have to use PAGE. Can blue native PAGE (BN-PAGE) solve this problem? I heard that in BN-PAGE the proteins will migrate according to their molecular size but still stay in native form.
It depends On how monomers are attached if it is a dimer at all. if they are attached by cystine bonds you still can perform SDS-PAGE. Otherwise it depends on what techniques are available to you.
No in PAGE proteins do not migrate according to their mass, completely false! they migrate according to their net charge and tertiary structure shape.
cheers,
DG
-DNA Gyrase-
QUOTE (DNA Gyrase @ Mar 2 2008, 04:08 PM)
QUOTE (The Bearer @ Mar 2 2008, 10:52 AM)
QUOTE (DNA Gyrase @ Mar 1 2008, 09:37 PM)
Easiest way is to run a SDS-PAGE. If you run a PAGE it won't give you any useful information about size of your protein. In SDS-PAGE proteins are denatured and are given a negative charge proportional to their mass.
I do not agree that PAGE give any useful information about size since in pI-appropriate native PAGE protein runs in retention to its size
What you mean? I didn't say PAGE, you said PAGE
QUOTE (The Bearer @ Feb 29 2008, 12:39 PM)
QUOTE (sarita @ Feb 29 2008, 04:39 AM)
How accurate can you estimate protein size from native gel? What means blue native gel compare to regular native gel?
it is reliable, however, to find the right running conditions, particularly the right buffer system, is tedious work;
I do not know the difference between blue and regulare native gel; go ask mdfenko
I said SDS-PAGE!
It is a standard method for determining size, so standard that you can find it in almost every biochemistry text book.
As I said PAGE won't give you any information about size. If you set pH to pI how do you suppose the protein is going to move in electric field? and decide do you want to set pH to pI of unknown protein or your marker?
take your words back or show me a reference.
Cheers,
DG
you wrote on 1st May: "If you run a PAGE it won't give you any useful information about size of your protein."
So, you did discus PAGE. If you think in terms of SDS-PAGE, it should be specified.
Running native PAGE with a pH of pI of the protein of interest actually does not make sense but I did not recommend this.
-The Bearer-
QUOTE (DNA Gyrase @ Mar 2 2008, 04:08 PM)
QUOTE (The Bearer @ Mar 2 2008, 10:52 AM)
QUOTE (DNA Gyrase @ Mar 1 2008, 09:37 PM)
Easiest way is to run a SDS-PAGE. If you run a PAGE it won't give you any useful information about size of your protein. In SDS-PAGE proteins are denatured and are given a negative charge proportional to their mass.
I do not agree that PAGE give any useful information about size since in pI-appropriate native PAGE protein runs in retention to its size
What you mean? I didn't say PAGE, you said PAGE
QUOTE (The Bearer @ Feb 29 2008, 12:39 PM)
QUOTE (sarita @ Feb 29 2008, 04:39 AM)
How accurate can you estimate protein size from native gel? What means blue native gel compare to regular native gel?
it is reliable, however, to find the right running conditions, particularly the right buffer system, is tedious work;
I do not know the difference between blue and regulare native gel; go ask mdfenko
I said SDS-PAGE!
It is a standard method for determining size, so standard that you can find it in almost every biochemistry text book.
As I said PAGE won't give you any information about size. If you set pH to pI how do you suppose the protein is going to move in electric field? and decide do you want to set pH to pI of unknown protein or your marker?
take your words back or show me a reference.
Cheers,
DG
you wrote on 1st May: "If you run a PAGE it won't give you any useful information about size of your protein."
So, you also did discus PAGE in general. If you thought in terms of SDS-PAGE, it should have been specified.
Running native PAGE with a pH of pI of the protein of interest actually does not make sense but I did not recommend this.
-The Bearer-
QUOTE
I heard that in BN-PAGE the proteins will migrate according to their molecular size but still stay in native form.
Unfortunately BN-PAGE is not that accurate in determining the size of a protein, this is because they migrate depending on how much coomassie they bind. Someone in your building must have an old FPLC laying around with a gel filtration column. Run some standards and that is probably the best method to determine molecular weight. HPLC is probably even more accurate but harder to come by.
-drewaight-
QUOTE (The Bearer @ Mar 3 2008, 10:00 AM)
QUOTE (DNA Gyrase @ Mar 2 2008, 04:08 PM)
QUOTE (The Bearer @ Mar 2 2008, 10:52 AM)
QUOTE (DNA Gyrase @ Mar 1 2008, 09:37 PM)
Easiest way is to run a SDS-PAGE. If you run a PAGE it won't give you any useful information about size of your protein. In SDS-PAGE proteins are denatured and are given a negative charge proportional to their mass.
I do not agree that PAGE give any useful information about size since in pI-appropriate native PAGE protein runs in retention to its size
What you mean? I didn't say PAGE, you said PAGE
QUOTE (The Bearer @ Feb 29 2008, 12:39 PM)
QUOTE (sarita @ Feb 29 2008, 04:39 AM)
How accurate can you estimate protein size from native gel? What means blue native gel compare to regular native gel?
it is reliable, however, to find the right running conditions, particularly the right buffer system, is tedious work;
I do not know the difference between blue and regulare native gel; go ask mdfenko
I said SDS-PAGE!
It is a standard method for determining size, so standard that you can find it in almost every biochemistry text book.
As I said PAGE won't give you any information about size. If you set pH to pI how do you suppose the protein is going to move in electric field? and decide do you want to set pH to pI of unknown protein or your marker?
take your words back or show me a reference.
Cheers,
DG
you wrote on 1st May: "If you run a PAGE it won't give you any useful information about size of your protein."
So, you did discus PAGE. If you think in terms of SDS-PAGE, it should be specified.
Running native PAGE with a pH of pI of the protein of interest actually does not make sense but I did not recommend this.
amazing explanation, I don't need to say anything readers can read quoted text and judge!
cheers,
DG
-DNA Gyrase-
Hello,
From the homepages of Invitrogen I found information of Invitrogen BN-PAGE system:
The NativePAGE™ Novex® Bis-Tris Gel System is a pre-cast polyacrylamide mini gel system that provides a sensitive and high-resolution method for analysis of native membrane protein complexes, native soluble proteins, molecular mass estimations, and assessment of purity of native proteins. It is based on the blue native polyacrylamide gel electrophoresis technique (BN PAGE) developed by Schagger and von Jagow (1-3).
And description of markers:
The NativeMark™ Unstained Protein Standard is a ready-to-use protein marker to allow for accurate molecular weight estimation of proteins using native gel electrophoresis with Tris-Glycine or NuPAGE® Novex Tris-Acetate Gels.
So from this description I get the idea that it is reliable for estimation of molecular weight. It is based in Coomassie and gradient gel
-sarita-
QUOTE (sarita @ Mar 5 2008, 08:50 AM)
Hello,
From the homepages of Invitrogen I found information of Invitrogen BN-PAGE system:
The NativePAGE™ Novex® Bis-Tris Gel System is a pre-cast polyacrylamide mini gel system that provides a sensitive and high-resolution method for analysis of native membrane protein complexes, native soluble proteins, molecular mass estimations, and assessment of purity of native proteins. It is based on the blue native polyacrylamide gel electrophoresis technique (BN PAGE) developed by Schagger and von Jagow (1-3).
And description of markers:
The NativeMark™ Unstained Protein Standard is a ready-to-use protein marker to allow for accurate molecular weight estimation of proteins using native gel electrophoresis with Tris-Glycine or NuPAGE® Novex Tris-Acetate Gels.
So from this description I get the idea that it is reliable for estimation of molecular weight. It is based in Coomassie and gradient gel
From the homepages of Invitrogen I found information of Invitrogen BN-PAGE system:
The NativePAGE™ Novex® Bis-Tris Gel System is a pre-cast polyacrylamide mini gel system that provides a sensitive and high-resolution method for analysis of native membrane protein complexes, native soluble proteins, molecular mass estimations, and assessment of purity of native proteins. It is based on the blue native polyacrylamide gel electrophoresis technique (BN PAGE) developed by Schagger and von Jagow (1-3).
And description of markers:
The NativeMark™ Unstained Protein Standard is a ready-to-use protein marker to allow for accurate molecular weight estimation of proteins using native gel electrophoresis with Tris-Glycine or NuPAGE® Novex Tris-Acetate Gels.
So from this description I get the idea that it is reliable for estimation of molecular weight. It is based in Coomassie and gradient gel
sarita, buddy,
I went to their website and read it but I really can't accept a quote from a website as a scientific reason they didn't said why and how PAGE is useful in determining molecular mass they just said it and maybe if you ask more informed persons within that company they tell you we meant this and we meant that.
If you refer me to a reference like a renown text book(stryer, voet,...) or a decent article(nature, JBC,...) I will take back my words with honor but the truth is all famous references are crying that PAGE won't give you any useful information about molecular mass and to the contrary of the website they present it with reasons that I indicated in previous posts.
cheers,
DG
-DNA Gyrase-
QUOTE (DNA Gyrase @ Mar 6 2008, 09:08 AM)
QUOTE (sarita @ Mar 5 2008, 08:50 AM)
Hello,
From the homepages of Invitrogen I found information of Invitrogen BN-PAGE system:
The NativePAGE™ Novex® Bis-Tris Gel System is a pre-cast polyacrylamide mini gel system that provides a sensitive and high-resolution method for analysis of native membrane protein complexes, native soluble proteins, molecular mass estimations, and assessment of purity of native proteins. It is based on the blue native polyacrylamide gel electrophoresis technique (BN PAGE) developed by Schagger and von Jagow (1-3).
And description of markers:
The NativeMark™ Unstained Protein Standard is a ready-to-use protein marker to allow for accurate molecular weight estimation of proteins using native gel electrophoresis with Tris-Glycine or NuPAGE® Novex Tris-Acetate Gels.
So from this description I get the idea that it is reliable for estimation of molecular weight. It is based in Coomassie and gradient gel
From the homepages of Invitrogen I found information of Invitrogen BN-PAGE system:
The NativePAGE™ Novex® Bis-Tris Gel System is a pre-cast polyacrylamide mini gel system that provides a sensitive and high-resolution method for analysis of native membrane protein complexes, native soluble proteins, molecular mass estimations, and assessment of purity of native proteins. It is based on the blue native polyacrylamide gel electrophoresis technique (BN PAGE) developed by Schagger and von Jagow (1-3).
And description of markers:
The NativeMark™ Unstained Protein Standard is a ready-to-use protein marker to allow for accurate molecular weight estimation of proteins using native gel electrophoresis with Tris-Glycine or NuPAGE® Novex Tris-Acetate Gels.
So from this description I get the idea that it is reliable for estimation of molecular weight. It is based in Coomassie and gradient gel
sarita, buddy,
I went to their website and read it but I really can't accept a quote from a website as a scientific reason they didn't said why and how PAGE is useful in determining molecular mass they just said it and maybe if you ask more informed persons within that company they tell you we meant this and we meant that.
If you refer me to a reference like a renown text book(stryer, voet,...) or a decent article(nature, JBC,...) I will take back my words with honor but the truth is all famous references are crying that PAGE won't give you any useful information about molecular mass and to the contrary of the website they present it with reasons that I indicated in previous posts.
cheers,
DG
the references you request are given on the web page from invitrogen:
invitrogen web page
make sure you click on "read all"
-mdfenko-
QUOTE (mdfenko @ Mar 7 2008, 11:40 AM)
QUOTE (DNA Gyrase @ Mar 6 2008, 09:08 AM)
QUOTE (sarita @ Mar 5 2008, 08:50 AM)
Hello,
From the homepages of Invitrogen I found information of Invitrogen BN-PAGE system:
The NativePAGE™ Novex® Bis-Tris Gel System is a pre-cast polyacrylamide mini gel system that provides a sensitive and high-resolution method for analysis of native membrane protein complexes, native soluble proteins, molecular mass estimations, and assessment of purity of native proteins. It is based on the blue native polyacrylamide gel electrophoresis technique (BN PAGE) developed by Schagger and von Jagow (1-3).
And description of markers:
The NativeMark™ Unstained Protein Standard is a ready-to-use protein marker to allow for accurate molecular weight estimation of proteins using native gel electrophoresis with Tris-Glycine or NuPAGE® Novex Tris-Acetate Gels.
So from this description I get the idea that it is reliable for estimation of molecular weight. It is based in Coomassie and gradient gel
From the homepages of Invitrogen I found information of Invitrogen BN-PAGE system:
The NativePAGE™ Novex® Bis-Tris Gel System is a pre-cast polyacrylamide mini gel system that provides a sensitive and high-resolution method for analysis of native membrane protein complexes, native soluble proteins, molecular mass estimations, and assessment of purity of native proteins. It is based on the blue native polyacrylamide gel electrophoresis technique (BN PAGE) developed by Schagger and von Jagow (1-3).
And description of markers:
The NativeMark™ Unstained Protein Standard is a ready-to-use protein marker to allow for accurate molecular weight estimation of proteins using native gel electrophoresis with Tris-Glycine or NuPAGE® Novex Tris-Acetate Gels.
So from this description I get the idea that it is reliable for estimation of molecular weight. It is based in Coomassie and gradient gel
sarita, buddy,
I went to their website and read it but I really can't accept a quote from a website as a scientific reason they didn't said why and how PAGE is useful in determining molecular mass they just said it and maybe if you ask more informed persons within that company they tell you we meant this and we meant that.
If you refer me to a reference like a renown text book(stryer, voet,...) or a decent article(nature, JBC,...) I will take back my words with honor but the truth is all famous references are crying that PAGE won't give you any useful information about molecular mass and to the contrary of the website they present it with reasons that I indicated in previous posts.
cheers,
DG
the references you request are given on the web page from invitrogen:
invitrogen web page
make sure you click on "read all"
hello, did you bother to read even abstract of those articles? They are talking about 2d electrophoresis and combining BN-PAGE with SDS-PAGE!
Abstract
Blue native Electrophoresis is a "charge shift" method developed for isolation of native membrane protein complexes from biological membranes that also separates both acidic and basic water-soluble proteins at a fixed pH of 7.5. In combination with a second dimension sodium dodecylsulfate electrophoresis it provides an analytical method for the determination of molecular mass and oligomeric state of nondissociated complexes, of subunit composition, and of degree of purity and for the detection of subcomplexes. The method was applied to analysis of cytochrome bc/bf complexes. By combination of a novel colorless native polyacrylamide gel electrophoresis (CN-PAGE) with blue native BN-PAGE, a two-dimensional native technique was developed that is suitable for preparation of highly pure membrane protein complexes.
As I had predicted if you ask more informed persons of the company they will say we meant this and we meant that, and we meant in combination with SDS-PAGE!
PAGE by itself won't give you any information about molecular mass! carve it in your brain! case closed!
cheers,
DG
-DNA Gyrase-
QUOTE (DNA Gyrase @ Mar 8 2008, 06:19 AM)
QUOTE (mdfenko @ Mar 7 2008, 11:40 AM)
QUOTE (DNA Gyrase @ Mar 6 2008, 09:08 AM)
QUOTE (sarita @ Mar 5 2008, 08:50 AM)
Hello,
From the homepages of Invitrogen I found information of Invitrogen BN-PAGE system:
The NativePAGE™ Novex® Bis-Tris Gel System is a pre-cast polyacrylamide mini gel system that provides a sensitive and high-resolution method for analysis of native membrane protein complexes, native soluble proteins, molecular mass estimations, and assessment of purity of native proteins. It is based on the blue native polyacrylamide gel electrophoresis technique (BN PAGE) developed by Schagger and von Jagow (1-3).
And description of markers:
The NativeMark™ Unstained Protein Standard is a ready-to-use protein marker to allow for accurate molecular weight estimation of proteins using native gel electrophoresis with Tris-Glycine or NuPAGE® Novex Tris-Acetate Gels.
So from this description I get the idea that it is reliable for estimation of molecular weight. It is based in Coomassie and gradient gel
From the homepages of Invitrogen I found information of Invitrogen BN-PAGE system:
The NativePAGE™ Novex® Bis-Tris Gel System is a pre-cast polyacrylamide mini gel system that provides a sensitive and high-resolution method for analysis of native membrane protein complexes, native soluble proteins, molecular mass estimations, and assessment of purity of native proteins. It is based on the blue native polyacrylamide gel electrophoresis technique (BN PAGE) developed by Schagger and von Jagow (1-3).
And description of markers:
The NativeMark™ Unstained Protein Standard is a ready-to-use protein marker to allow for accurate molecular weight estimation of proteins using native gel electrophoresis with Tris-Glycine or NuPAGE® Novex Tris-Acetate Gels.
So from this description I get the idea that it is reliable for estimation of molecular weight. It is based in Coomassie and gradient gel
sarita, buddy,
I went to their website and read it but I really can't accept a quote from a website as a scientific reason they didn't said why and how PAGE is useful in determining molecular mass they just said it and maybe if you ask more informed persons within that company they tell you we meant this and we meant that.
If you refer me to a reference like a renown text book(stryer, voet,...) or a decent article(nature, JBC,...) I will take back my words with honor but the truth is all famous references are crying that PAGE won't give you any useful information about molecular mass and to the contrary of the website they present it with reasons that I indicated in previous posts.
cheers,
DG
the references you request are given on the web page from invitrogen:
invitrogen web page
make sure you click on "read all"
hello, did you bother to read even abstract of those articles? They are talking about 2d electrophoresis and combining BN-PAGE with SDS-PAGE!
Abstract
Blue native Electrophoresis is a "charge shift" method developed for isolation of native membrane protein complexes from biological membranes that also separates both acidic and basic water-soluble proteins at a fixed pH of 7.5. In combination with a second dimension sodium dodecylsulfate electrophoresis it provides an analytical method for the determination of molecular mass and oligomeric state of nondissociated complexes, of subunit composition, and of degree of purity and for the detection of subcomplexes. The method was applied to analysis of cytochrome bc/bf complexes. By combination of a novel colorless native polyacrylamide gel electrophoresis (CN-PAGE) with blue native BN-PAGE, a two-dimensional native technique was developed that is suitable for preparation of highly pure membrane protein complexes.
As I had predicted if you ask more informed persons of the company they will say we meant this and we meant that, and we meant in combination with SDS-PAGE!
PAGE by itself won't give you any information about molecular mass! carve it in your brain! case closed!
cheers,
DG
now, if you read the paper (1991) (and more specifically, the discussion) you would have seen that the author was claiming to make some estimate of size. the second dimension was to see the subunits of the complexes being studied.
even native gels can give some size estimation (which can be enhanced with a gradient). the gel matrix and porosity will allow smaller proteins to pass through faster than large proteins with the same or similar charge.
here is an excerpt from invitrogen:
Nondenaturing or “native” electrophoresis (i.e., electrophoresis in the absence of denaturants such as detergents and urea) is an often-overlooked technique for determining the native size, subunit structure, and optimal separation of a protein. Because mobility depends on the size, shape, and intrinsic charge of the protein, nondenaturing electrophoresis provides a set of separation parameters distinctly different from mainly size-dependent denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and charge-dependent isoelectric focusing.
'nuff said.
-mdfenko-