ENCAB000AJY

Antibody against Homo sapiens PITX1

Homo sapiens
at least one cell type or tissue
awaiting characterization
Status
released
Source (vendor)
Santa Cruz Biotech
Product ID
sc-81979
Lot ID
J0109
Characterized targets
PITX1 (Homo sapiens)
Host
mouse
Clonality
monoclonal
Isotype
IgG
Antigen description
Raised against recombinant Pitx1 of human origin.

Characterizations

PITX1 (Homo sapiens)
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_PITX1_sc81979_09122011_MassSpec.pdf), including common contaminants. Target protein is listed as hit 7 in ~45 kDa band. ENCODE data standards recognizes various methodologies for secondary validation of antibodies. Among these methodologies is immunoprecipitation followed by mass spectrometry analysis. 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 Coomasie Blue in order to visualize marker bands. A gel fragment corresponding to the band indicated above in the western blot image was excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. There the sample was run on an LTQ XL Linear Ion Trap Mass Spectrometer with alternating collision-induced dissociation and electron-transfer dissociation. Peptides were identified using MASCOT (Matrix Science), with probability based matching at p < 0.05. Subsequent analysis was performed in Scaffold (Proteome Software, Inc.) at 0.0% protein FDR and 0.3% peptide FDR. As per ENCODE data standards, all Scaffold results are listed below, including common contaminants. Target protein is highlighted in bold font.
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
PITX1 (Homo sapiens)
Method: immunoprecipitation
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-81979 in whole cell lysate (WCL) of K562 and HeLa; PM=protein marker. PITX1 band is indicated.
Submitted by
Richard Myers
Lab
Richard Myers, HAIB