Anti-PAF1 antibody - ChIP Grade (ab20662)

  Western blot - PAF1 antibody (ab20662)

All lanes : Anti-PAF1 antibody - ChIP Grade (ab20662) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with PAF1 peptide (ab23389) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG (Alexa Fluor® 680) (ab28446) at 1/10000 dilution

Predicted band size : 60 kDa
Observed band size : 85 kDa (why is the actual band size different from the predicted?)
Additional bands at : 73 kDa (possible cleavage fragment,cross reactivity).

ab20662 recognizes a band at approximately 80kDa, which corresponds in size to PAF1. Although it has a calculated molecular weight of ~60kDa, several groups have shown that it migrates at a size of 80-90kDa (see Moniaux, et.al., Oncogene Feb. 2006, Yart et.al., Mol Cell Biol 25:5052-60, 2005, and Rozenblatt-Rosen, et.al., Mol Cell Biol 25:612-20, 2005). There is also a smaller band at ~73kDa which may be a degradation product. Both bands are competed away by the addition of immunizing peptide, suggesting that the interaction is specific.

  ChIP - PAF1 antibody (ab20662)

Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 5µg of  ab20662 (blue), and 20µl of Protein A/G sepharose beads in RIPA buffer. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the GAPDH (active) and MYO-D and SERPINA (inactive) promoters and over the y-Actin gene (active). Schematic diagram of the y-Actin gene is shown on the top of the figure. Black boxes represent exons and thin lines represent introns. PCR products are depicted as bars under the gene.