"michelin" at ohsu.edu.geibels
Thu Jan 31 11:37:13 EST 2002
I have a problem with an IP protocol, it is similar to one others have
described. My protein of interest is a large heterodimeric membrane
protein with a MW of ~105kDa/~37kDa and once Ip'd the large subunit runs
right where the dimeric heavy chain Ig runs (~100kDa) thus obscuring the
2ndary detection. Many have suggested nonreducing gels but there is
always some Ab at 100kDa and I would like to get rid of it.
It seems to me that COMPLETE reduction of the Ab should give only 50kDa
(heavy chain) and 25kDa (light chain) bands but I haven't found
conditions to do this yet. Is it possible?
I have one ref that TCEP at 15uM at RT would do it but 1mM doesn't in my
hands. A complication is that the large subunit of my protein can't be
boiled, it vanishes from SDS PAGE gels if heated too high (?!) this is
supposedly not rare for large membrane proteins.
I'm exploring temps and TCEP/DTT/BME conc. but haven't found the answer.
Am I naive? Are Ab never fully reduced without boiling? Whay haven't
others suggested complete Ab reduction over non reducing gels?
Any insight would be appreciated, and if I find conditions that work I
will pass them on.
michelin at ohsu.edu
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