1. Antibodies
  2. Homo sapiens
  3. CEBPD

ENCAB000AFC

Antibody against Homo sapiens CEBPD

Homo sapiens
HepG2, HeLa-S3, liver, K562, MCF-7
not characterized to standards
Status
released
Source (vendor)
Santa Cruz Biotech
Product ID
sc-636
Lot ID
C1010
Characterized targets
CEBPD (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Isotype
IgG
Antigen description
Epitope mapping at the C-terminus of CEBPD of human origin.

Characterizations

CEBPD (Homo sapiens)
K562HepG2MCF-7
Method: immunoprecipitation
Attachment from submitter
Attachment from submitters
not compliant
Caption
Whole cell lysates of K562, MCF7, and HepG2 were immunoprecipitated using the primary antibody (Santa Cruz Biotechnology; sc-636). The IP fraction was separated on a 12% acrylamide gel with the Bio-Rad PROTEAN II xi system. After separation, the samples were transferred to a nitrocellulose membrane with an Invitrogen iBlot system. The membrane was probed with the primary antibody (same as that used for IP) and a secondary HRP-conjugated antibody. The resulting bands were visualized with SuperSignal West Femto Solution (Thermo Scientific). Protein Marker (PM) is labeled in kDa. The approximate size of CEBPD is ~28 kDa.
Reviewer comment
Band of interest is not 50% of overall signal in lane
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
CEBPD (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment PDF Icon
not compliant
Caption
IP followed by mass spectrometry: Briefly, HeLa 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_CEBPD_(SC-636)_07192011_MassSpec.pdf), including common contaminants. Target protein is listed as hit 28 in lower (~35 kDa) band and not detected in upper (~120 + kDa) bands. No proteins with known DNA-binding activity were detected in the upper bands.
Reviewer comment
Not within top 20 factors ranked by fold enrichment. Proper document of results not provided.
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
CEBPD (Homo sapiens)
HepG2HeLa-S3
Method: immunoblot
Attachment from submitter
Attachment from submitters
not compliant
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. Upper bands also visualized at ~120 kD. Bands at ~35 kD and ~120+ kD tested by IP-mass spec. Only the band at ~35 kD was positive for CEBPD. Figure legend: IP-western with sc-636 in whole cell lysate (WCL) of HepG2 and HeLa; PM=protein marker. CEBPD bands are indicated. Expected size: ~28.5 kDa.
Reviewer comment
HepG2: No band visible in the expected size range. HeLa-S3: Band is not at least 50% of overall lane signal
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
CEBPD (Homo sapiens)
liver
Method: immunoblot
Attachment from submitter
Attachment from submitters
not compliant
Caption
The ENCODE Binding Working Group finds for some valuable tissues that recreating a primary on well characterized antibodies is not cost effective. Therefore, they allow exemption from standards for these tissues.
Submitter comment
The lab is asking for an exemption for liver cells due to the lack of resource to make a primary characterization for them.
Reviewer comment
Not compliant due to a lack of at least two compliant characterizations in other tissue/cell types.
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
Download
 No_tissue.png