Recombinant Antibody Production -Reducing / Non-reducing SDS page conditions - (Oct/20/2010 )
Hi Guys,
I have a strange problem and I was curious if any of you protein gurus out there might have an explanation. We're working on producing a Human Fc tagged recombinant ScFv monoclonal antibody. When we started doing transient transfections into Cho-K1 mammalian cells we saw both our antibody monomer (56 kda) and dimer (112 kda) on reducing (with dtt/heating) and non-reducing (no dtt/no heating) sds page gels at equal intensity (in june). We stopped working for it for a number of months but continued to passage and create a stable cell line which had the same charectoristics for the monomer and dimer (last tested under selective pressure in August, and saw the same as with the transient transfection). The cells were continually maintained under selective pressure since we knew we would revisit them. When we finally went back to re examine them we have been noticing that under reducing conditions the monomer has become much weaker maybe 1/10 the intensity of the non-reduced antibody whereas before they where equal intensity when loaded at the same concentration. At first we thought it was a technical issue, but after multiple western blots all showing the same trend we're at a bit of a loss. It appears that heat denaturing and dtt are now destroying the protein completely. Functionally the secreted antibody appears to be performing the same and will still bind its target sequence, however it appears to be much more fragile than it was initially. Does anyone have an explanation?
We're planning on going back to some frozen stocks from when we tested in August, however my concern is that in a few passages the same thing will start occuring again. It doesnt seem to have any functional significance (however we have not done any half-life studies on it yet). Any ideas what could be going on? Oh and also, mRNA expression and genomic expression of the integrated plasmid sequence is still there.
Thanks!
Have you looked into aggregation (% - of total protein), non specific binding due to thermally induced refolding in the non reduced step when setting up your gel. Maybe what im getting at is that it could be an assay issue and that the cells are fine. Can i ask if you are using slab gels or capillary electrophoresis?
Cheers
sbyrne27
cancergeek on Wed Oct 20 18:43:14 2010 said:
Hi Guys,
I have a strange problem and I was curious if any of you protein gurus out there might have an explanation. We're working on producing a Human Fc tagged recombinant ScFv monoclonal antibody. When we started doing transient transfections into Cho-K1 mammalian cells we saw both our antibody monomer (56 kda) and dimer (112 kda) on reducing (with dtt/heating) and non-reducing (no dtt/no heating) sds page gels at equal intensity (in june). We stopped working for it for a number of months but continued to passage and create a stable cell line which had the same charectoristics for the monomer and dimer (last tested under selective pressure in August, and saw the same as with the transient transfection). The cells were continually maintained under selective pressure since we knew we would revisit them. When we finally went back to re examine them we have been noticing that under reducing conditions the monomer has become much weaker maybe 1/10 the intensity of the non-reduced antibody whereas before they where equal intensity when loaded at the same concentration. At first we thought it was a technical issue, but after multiple western blots all showing the same trend we're at a bit of a loss. It appears that heat denaturing and dtt are now destroying the protein completely. Functionally the secreted antibody appears to be performing the same and will still bind its target sequence, however it appears to be much more fragile than it was initially. Does anyone have an explanation?
We're planning on going back to some frozen stocks from when we tested in August, however my concern is that in a few passages the same thing will start occuring again. It doesnt seem to have any functional significance (however we have not done any half-life studies on it yet). Any ideas what could be going on? Oh and also, mRNA expression and genomic expression of the integrated plasmid sequence is still there.
Thanks!
Hola, I think that loading buffer has lost its reducer capacity.Add 1ul of concentrated mercaptoethanol to each sample before boil and if the relation monomer/dimer continues has to look for other explanation. Buena suerte