F(ab) mystery

[Pamela Norton]

In article <3CF24954.7538E8B3 at mcgill.ca>, Allison < THIS"@mcgill.ca>
wrote:

> We are currently doing papain digests of monoclonal antibodies in order
> to make F(ab) fragments.  In a recent experiment we have noticed
> something strange when running non-reducing SDS-PAGE to check the final
> product (after digestion and cleanup on a Protein A column).  In the
> lane with 0.5 uL of the F(ab) we see a strong band at about 45K as
> expected.  In the lane with 1.0 uL of the F(ab) the 45K band is weaker
> and we see a strong band of about 30-32K.  The only difference being the
> amount loaded on the gel.
> 
> We have seen this 30K band before but much weaker.  There is no obvious
> correlation with the type of Mab used to make the F(ab), or with the
> final concentration of the F(ab) prep.
> 
> Any ideas?
> 
> Allison

It sounds as though you might be getting some degree of reduction. Is
the 1.0 ul lane next to a marker lane or something else that could be
diffusing over?

Just a guess.

Pam