ENCAB000ALE

Antibody against Mus musculus SRF, Homo sapiens SRF

Mus musculus
at least one cell type or tissue
awaiting characterization
Homo sapiens
at least one cell type or tissue
Status
released
Source (vendor)
Santa Cruz Biotech
Product ID
sc-335
Lot ID
F3006
Lot ID aliases
H2604, L2005
Host
rabbit
Clonality
polyclonal

Characterizations

SRF (Mus musculus)
Method: immunoprecipitation
Attachment from submitter
not reviewed
Caption
Whole lysate from C2C12 cells was immunoprecipitated with the SRF (Santa Cruz Biotech, sc-335) antibody. 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. The membrane was then blotted with primary antibody (same as that used for IP) and then a secondary HRP-conjugated antibody. The resulting bands were visualized using SuperSignal West Femto solution (Thermo Scientific). A band of expected size for the SRF target (~40 kD) was detected, along with contamination from the heavy chain (~50 kD) and light chain (~25kD) of the primary antibody used for IP.
Submitted by
Flo Pauli-Behn
Lab
Richard Myers, HAIB
SRF (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-335 in whole cell lysate (WCL) of K562, HepG2, GM12878 and HeLa; PM=protein marker. SRF bands are indicated.
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
SRF (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_SRF_sc335_09122011_MassSpec.pdf), including common contaminants. Target protein is listed as hit 26 in ~65 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 0.0% 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