Free HRP in HRP conjugated IgG MAb

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antibodix
antibodix's picture
Free HRP in HRP conjugated IgG MAb

I am currently working on developing an In-House Protocol for determining free HRP in HRP Conjugated MAb, Surprisingly I am having great difficulty in separating HRP and IgG on a SEC column(Just seems silly, but I just can't), I use Agilent 1100 lc, SEC 5um particle 300A Pore Size column, I use Mobile Phase PBS 7.4(10mM) in 138mM Nacl
Should i try a C18 column and run a RP chromotography with a gradient?
What do you guys recommend, also i have seen some salt deposits on my flow cell, i cleaned it, but do you guys recomment to run IPA or IPA+WATER through the system (without the coumn) to clean the flow cell??
Please help me

Dr. Analytical
Dr. Analytical's picture
We need more information

We need more information about your SEC system, especially the column that you used.  Remember, SEC is not a high resolution technique, and you must choose the proper MW range of the column.  Exactly what problems are you having?

There are reversed phase methods for antibodies, but they are sometimes difficult to reproduce.

Any salt deposits on an LC are a bad sign.  It means you have a leak, and you need to fix the leak before you do anything else.

efedaloglu
efedaloglu's picture
you try a dialysis

you try a dialysis