rabbit anti-ptpn13 nb100-56139  (Novus Biologicals)

Journal: Scientific Reports

Article Title: Proteins interacting with cloning scars: a source of false positive protein-protein interactions

doi: 10.1038/srep08530

Figure Lengend Snippet: (A), a region within the C-terminus of Flexi cloned TNIP2 is important for its association with PTPN13. Plasmids expressing the six Halo-tagged constructs indicated were transiently transfected in HEK293T cells for Halo affinity purification followed by MudPIT mass spectrometry analysis. Relative amounts of the five prey proteins indicated in enriched using each of these six baits (FDR < 0.05) are indicated according to their relative dNSAF value. Average prey dNSAF values were calculated from between three and six replicate experiments for each bait (see ). Average prey dNSAF values were then normalized to the average bait dNSAF to generate relative dNSAF values. (B), the association of Flexi-cloned TNIP2 and PTPN13 depends on the C-terminal valine cloning scar. Whole cell extracts from HEK293T cells transfected with the indicated constructs were subjected to Halo affinity chromatography and samples were analysed by SDS-PAGE followed by Western blotting. TNIP2 protein was visualized using rabbit anti-TNIP2 or rabbit anti-PTPN13 primary antibodies, and Alexa-680 labeled anti-rabbit secondary antibodies. Note the change in molecular weight of the TNIP2 bait after purification, which involves removal of the 33 kDa Halo tag. Western blots were imaged using a Li-Cor infra-red imaging system. (C), Halo purified proteins from HEK293T cells transfected with Halo-TNIP2 253–429 (5 biological replicates), Halo-TNIP2 253–429 no valine (3 biological replicates) and Halo-PTPN13 (3 biological replicates) were analysed by mass spectrometry. The numbers of distributed MS/MS spectra corresponding to the proteins TNIP2, PTPN13 and STXBP4 from each of these replicates is indicated. None of these proteins were detected in 9 biological replicates of control purifcations using cells expressing the Halo tag alone.

Article Snippet: Rabbit anti-TNIP2 (NBP1-32689) and rabbit anti-PTPN13 (NB100-56139) polyclonal antibodies were from Novus Biologicals.

Techniques: Clone Assay, Expressing, Construct, Transfection, Affinity Purification, Mass Spectrometry, Cloning, Affinity Chromatography, SDS Page, Western Blot, Labeling, Molecular Weight, Purification, Imaging, Tandem Mass Spectroscopy, Control

Journal: Scientific Reports

Article Title: Proteins interacting with cloning scars: a source of false positive protein-protein interactions

doi: 10.1038/srep08530

Figure Lengend Snippet: Part of the PTPN13 ORF coding for a 903 aa region, which included the five PDZ domains, was subcloned into FLAG-pcDNA5/FRT and coexpressed in HEK293T cells with or without Halo-TNIP2 (with the valine cloning scar) as indicated. Lysates were subjected to Halo affinity purification and the resulting eluates analysed by SDS-PAGE and Western blotting. Proteins were detected using anti-FLAG® M2 mouse monoclonal and anti-TNIP2 rabbit polyclonal primary antibodies, and with IRDye® 680LT labeled anti-mouse (red) and IRDye® 800CW anti-rabbit (green) secondary antibodies. Proteins were visualized using a LI-COR® Odyssey® infrared imaging system.

Article Snippet: Rabbit anti-TNIP2 (NBP1-32689) and rabbit anti-PTPN13 (NB100-56139) polyclonal antibodies were from Novus Biologicals.

Techniques: Cloning, Affinity Purification, SDS Page, Western Blot, Labeling, Imaging