Transcript IgGbindingcall.pptx

Incorporation of IgG for the antibody validation for
RNA Binding Proteins (RBP)
Balaji Sundararaman
Yeo Lab
UCSD.
 Yeo lab has validated about 700 antibodies against RBP for IP-WB assay.
But, the validation did not had IgG control IP experiments.
 Lot information for 611 antibodies were registered with DCC, of which 411
are under ‘awaiting lab characterization’ and 200 are in ‘not pursued’
category.
 About 320 antibodies (250+70 as backup) are prioritized for the 250 RBP
CLIPseq assay production goal.
 The proposal is to perform IgG control IP experiments for the 320
antibodies, during the CLIP experiments using the same antibodies.
 Images for primary validation either have to be electronically trimmed or
must be linked with each CLIP experiment in the DCC site.
Fig 1
Fig 2
Figure Legend:
Fig 1: IP-WB with 2.5% Input (I), 2.5% Supernatant after IP (S) and 10% IP Bead enrichment (B) for
Replicate 1, High Rnase control and no-UV control samples from a CLIP experiment and 2.5% Input (I)
and 50% IP Bead enrichment (B) for IgG IP experiments. The no-UV control and IgG control lanes would
be considered as primary validation for antibody , rest of the lanes are considered as QC for CLIP.
Fig 2: Final image with 2.5% Input (I), 2.5% Supernatant after IP (S) and 10% IP Bead enrichment (B) for
Replicate 1 and no-UV control of a CLIP experiment run along with Input, Sup, Bead samples from old
antibody IP validation experiment along with Input and Bead samples for IgG control.