Expect monomer on SDS PAGE gel get dimer or larger

hi all,
have a bit of crisis here with a protein which contains 3 cysteines. in humans and other organisms a monomer can be obtained on a SDS PAGE gel but for my protein a get larger aggregates. also i have checked predicted MW and this gives a theoretical monomer also. i have tried to add THP (a reducer recommended by merck biosciences) to HIS tag purification. Also have added B mercaptoethanol and DTT. all in varying amounts and for various times with no avail. i presumed when this did not work that i just needed to purify under denaturing conditions but this has not worked either. i used 8M urea, sodium phosphate and TrisCl for this. has anyone any ideas i really need to get this working ASAP!
thanks

QUOTE (bridgetc @ Jun 20 2007, 10:18 AM)

hi all,
have a bit of crisis here with a protein which contains 3 cysteines. in humans and other organisms a monomer can be obtained on a SDS PAGE gel but for my protein a get larger aggregates. also i have checked predicted MW and this gives a theoretical monomer also. i have tried to add THP (a reducer recommended by merck biosciences) to HIS tag purification. Also have added B mercaptoethanol and DTT. all in varying amounts and for various times with no avail. i presumed when this did not work that i just needed to purify under denaturing conditions but this has not worked either. i used 8M urea, sodium phosphate and TrisCl for this. has anyone any ideas i really need to get this working ASAP!
thanks

first, there is to distinguish between aggregation and (self-)assembly; both have physiological function;

among others, phosphorylations can direct assembly as well as aggregation; aggregation may involve other proteins...

so, you have to check, if it is aggregation or assemby, and second, what are the conditions;

during prepation, especially, aggregation can be induced, f.i. if protein solution is more concentrated than in physiological systems

QUOTE (The Bearer @ Jun 20 2007, 09:59 AM)

QUOTE (bridgetc @ Jun 20 2007, 10:18 AM)

hi all,
have a bit of crisis here with a protein which contains 3 cysteines. in humans and other organisms a monomer can be obtained on a SDS PAGE gel but for my protein a get larger aggregates. also i have checked predicted MW and this gives a theoretical monomer also. i have tried to add THP (a reducer recommended by merck biosciences) to HIS tag purification. Also have added B mercaptoethanol and DTT. all in varying amounts and for various times with no avail. i presumed when this did not work that i just needed to purify under denaturing conditions but this has not worked either. i used 8M urea, sodium phosphate and TrisCl for this. has anyone any ideas i really need to get this working ASAP!
thanks

first, there is to distinguish between aggregation and (self-)assembly; both have physiological function;

among others, phosphorylations can direct assembly as well as aggregation; aggregation may involve other proteins...

so, you have to check, if it is aggregation or assemby, and second, what are the conditions;

during prepation, especially, aggregation can be induced, f.i. if protein solution is more concentrated than in physiological systems

thanks bearer
i'm a newbie to protein biochem so i have no idea how to tell the difference between the two. can you direct me to any papers or protocols which may help?http://www.protocol-online.org/forums/style_images/1/folder_post_icons/icon9.gif
http://www.protocol-online.org/forums/styl...icons/icon9.gif
thank you very much!

Expect monomer on SDS PAGE gel get dimer or larger - (Jun/20/2007 )