ENCAB000ALI

Homo sapiens

at least one cell type or tissue

awaiting characterization

Method: immunoprecipitation followed by mass spectrometry

not reviewed

Caption: IP followed by mass spectrometry: Briefly, K562 whole cell lysates were immunoprecipitated using primary antibody, and the IP fraction was loaded on a 12% acrylamide gel and separated with a Bio-Rad PROTEAN II xi system. Gel was stained with Coomassie Blue in order to visualize marker bands. Gel fragments corresponding to the bands indicated above in the western blot image were excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. There the samples were run on an LTQ XL Linear Ion Trap Mass Spectrometer by LC-ESI-MS/MS. Peptides were identified using SEQUEST tandem mass spectra analysis, with probability based matching at p < 0.05. As per ENCODE data standards, all SEQUEST results are attached (ENCODE_HAIB_YYY_sc77742_09262011_MassSpec.pdf), including common contaminants. Target protein is listed as hit 2a in the ~90kDa band.
Submitted by: Richard Myers
Lab: Richard Myers, HAIB
Grant: U54HG004576
Download: human_STAT5A_SC-74442_validation_Myers.pdf

Method: immunoblot

not reviewed

Caption: Western blot protocol: Whole cell lysate was immunoprecipitated using primary antibody, and the IP fraction was loaded on a 12% acrylamide gel and separated with a Bio-Rad PROTEAN II xi system. After separation, the samples were transferred to a nitrocellulose membrane with an Invitrogen iBlot system. Blotting with primary (same as that used for IP) and secondary HRP-conjugated antibodies was performed on an Invitrogen BenchPro 4100 system. Visualization was achieved using SuperSignal West Femto solution (Thermo Scientific). Results: Band of expected size visualized, representing strongest signal in the lane. Figure legend: IP-western with sc-77742 in whole cell lysate (WCL) of K562 and GM12878; PM=protein marker. STAT5A bands are indicated.
Submitted by: Richard Myers
Lab: Richard Myers, HAIB
Grant: U54HG004576
Download: human_STAT5A_SC-74442_validation_Myers.pdf