2 replies to this topic

[Dhananjaya Dendukuri]

Hello all,

I have been long struggling with reducing the sulfhydryl groups of an IgG.  I am using a 25 mM solution of MEA (mercaptoethyl amine) to reduce a 2 mg/ml solution of IgG in the presence of EDTA.  

The source of my problems seems to be the reconjugation of cleaved IgG back into its original form. I have tried various solutions including a de-aerated environment to prevent oxidation but to no avail    Does anyone have a detailed protocol for this reduction?

Best,
D

[rick112]

hi
could i ask you for what you want to reduce IgG???
i have used following protocol...hope this helps...it gives me good results....

take 20 ul of 10mg/ml of IgG..to this add 4 ul of DTT from a stock of 0.25 M followed by 152 ul of Tris-GuHCL (6M) pH 7.47.....incubate at 75oC for 5 min.....add 12 ul of IAA from a stock of 0.25M..incubate at dark for 40 min in R.T....to stop the reaction add 12 ul of 0.25M DTT

hope this help

[Dhananjaya Dendukuri]

Thank you for your reply Rick.  I am trying to selectively reduce the disulfide bonds in the hinge region before performing a sulfyhydryl-maleimide conjugation.  The aim is to conjugate the IgG with a fluorophore and not to completely reduce the protein.  I need my antibody in the native state because it will be used for an immunoassay after conjugation.   Let me know if you have any thoughts about this.

rick112, on Jan 28 2010, 01:27 PM, said:

hi
could i ask you for what you want to reduce IgG???
i have used following protocol...hope this helps...it gives me good results....

take 20 ul of 10mg/ml of IgG..to this add 4 ul of DTT from a stock of 0.25 M followed by 152 ul of Tris-GuHCL (6M) pH 7.47.....incubate at 75oC for 5 min.....add 12 ul of IAA from a stock of 0.25M..incubate at dark for 40 min in R.T....to stop the reaction add 12 ul of 0.25M DTT

hope this help