Hello,
I have purified a monoclonal antibody from an hybridoma supernatant. When I run different eluted fractions on an SDS-PAGE, I get only one band in reducing condition (the 150 kDa band). Before being loaded on the gel, samples were boiled in the presence of 2-mercaptoethanol 5 min at 95 °C. To prepare the sample buffer, I followed the instructions from the manufacturer (add reducing agent : 100 µL 2-mercaptoethanol per 900 µL 4x Laemmli buffer; then dilute sample : 3 parts sample with 1 part sample buffer).
I was thinking of maybe using a "stronger" reducing agent like DTT?
I've attached a picture of the gel. Well 2 : Ab sample non reduced, Well 3 to 7 : different Ab sample in reduced condition.
I'm also confused by the band at 75 kDa...
Can anyone please help me to figure it out?
Thanks in advance.