7 replies to this topic

[medchemgirl]

If I wanted to detect a protein that normally would be 9kDa as a monomer, and I got it biotinylated, would it be detected in a WB at a different molecular weight using Streptavidin-HRP for detection? And how much more heavier would it be?

[Gerard]

MW biotin 244g,  your protein MW 9000g

You can,t tell the difference in the blot

[medchemgirl]

Ohh, I see, so I shouldn't worry about it then.

Gerard, on Nov 12 2009, 11:26 AM, said:

MW biotin 244g,  your protein MW 9000g

You can,t tell the difference in the blot

[mdfenko]

it may be relatively insignificant if there is only one biotin bound per protein molecule but if more biotin binds then the effect will become more significant.

also, which page formulation do you use? if you are using tris-tricine then one biotin may make a significant difference on the gel.

[Feelcontraire]

medchemgirl, on Nov 12 2009, 07:44 AM, said:

If I wanted to detect a protein that normally would be 9kDa as a monomer, and I got it biotinylated, would it be detected in a WB at a different molecular weight using Streptavidin-HRP for detection? And how much more heavier would it be?

You may observe differences higher than expected as biotin is a vitamin that may lead to a different ratio of weight/mobility than that of aminoacids. This is due to it's conductivity, hydrophobicity and SDS interacting characteristics.

[medchemgirl]

I was actually just reading about the Tris-Tricine gels. I have never used them. I am using Nupage Bis-Tris gel from Invitrogen.

mdfenko, on Nov 13 2009, 02:32 PM, said:

it may be relatively insignificant if there is only one biotin bound per protein molecule but if more biotin binds then the effect will become more significant.

also, which page formulation do you use? if you are using tris-tricine then one biotin may make a significant difference on the gel.

[Feelcontraire]

medchemgirl, on Nov 19 2009, 12:06 PM, said:

I was actually just reading about the Tris-Tricine gels. I have never used them. I am using Nupage Bis-Tris gel from Invitrogen.

mdfenko, on Nov 13 2009, 02:32 PM, said:

it may be relatively insignificant if there is only one biotin bound per protein molecule but if more biotin binds then the effect will become more significant.

also, which page formulation do you use? if you are using tris-tricine then one biotin may make a significant difference on the gel.

You can use your Nupage Bis-Tris gels with MES instead of MOPS or HEPES which will give a similar result as tricine.

Search for MES monohydrate it's much cheaper than just MES.

[medchemgirl]

That is what I'm using actually, but the MES, not the monohydrate. I will check that.  Thanks

Feelcontraire, on Nov 19 2009, 07:47 PM, said:

medchemgirl, on Nov 19 2009, 12:06 PM, said:

I was actually just reading about the Tris-Tricine gels. I have never used them. I am using Nupage Bis-Tris gel from Invitrogen.

mdfenko, on Nov 13 2009, 02:32 PM, said:

it may be relatively insignificant if there is only one biotin bound per protein molecule but if more biotin binds then the effect will become more significant.

also, which page formulation do you use? if you are using tris-tricine then one biotin may make a significant difference on the gel.

You can use your Nupage Bis-Tris gels with MES instead of MOPS or HEPES which will give a similar result as tricine.

Search for MES monohydrate it's much cheaper than just MES.