Hi All, Thanks to all that replied for the advice, suggestions etc. about my antibody. I will definitely try the suggestions. Christine
________________________________ From: Sheemei Lok [mailto:sheeme...@yahoo.com.sg] Sent: Monday, May 17, 2010 9:56 PM To: Harman, Christine Subject: Re: [ccp4bb] Antibody digest with Ficin Hi, I included 1mM DTT in the buffer and it got rid of the non-specific disulphide bridges. sheemei ________________________________ From: "Harman, Christine" <christine.har...@fda.hhs.gov> To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, 17 May 2010 15:37:54 Subject: [ccp4bb] Antibody digest with Ficin Hi All, I figured there are plenty of Fab/peptide structures out there that I may find a crystallographer/antibody expert out there who can give me some advice. I am interested in obtaining a mFab/peptide complex structure. I have reached the stage where I have successfully produced Fab from mouse IgG1 using immobilized ficin (Kit from Pierce). After ficin digest I purify the Fab with protein A column ( included in kit from Pierce) and it appears relatively pure with minor contamination of F(ab')2 and possibly some minor amount of fragmented Fc. I planned on doing IEC or SEC but FPLC was down and couldn't proceed to next step immediately. I concentrated/buffer exchanged my Fab fraction in PBS and stored at 4ºC over the weekend. The problem arose when I ran a gel I noticed that my Fab fraction which previously (sampled before diafiltration) ran ~50kDa band was now running as small smear around 100kDa. I have a suspicion that the concentrating and resuspending (for buffer exchange) may have some role and what might have happened could be related to possibly non-specific disulfide formation; however, was hoping someone may have some ideas or may have experienced this before. Given my antibody subtype being G1, I used ficin as opposed to papain and my experience is limited in knowing the types of fragments that are generated with ficin. Pierce claims their ficin kit produces Fab and F(ab')2; however, I was wondering if ficin may also produce Fab' given that at different concentrations of cysteine either Fab or F(ab')2 can be generated. Any thoughts, comments or ideas would be much appreciated. Much Thanks, Christine Harman
