Revision 3

#83932Store at -20C

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP, IF-IC, FC-FP, ChIP, C&R

REACTIVITY:

M

SENSITIVITY:

Endogenous

MW (kDa):

45

Source/Isotype:

Rabbit IgG

UniProt ID:

#P20263

Entrez-Gene Id:

18999

Product Information

Product Usage Information

For optimal ChIP results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100
Immunofluorescence (Immunocytochemistry) 1:800
Flow Cytometry (Fixed/Permeabilized) 1:200
Chromatin IP 1:50
CUT&RUN 1:50

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

Oct-4A (D6C8T) Rabbit mAb recognizes endogenous levels of total murine Oct-4A protein.

Species Reactivity:

Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val80 of murine Oct-4A protein.

Background

Oct-4 (POU5F1) is a transcription factor highly expressed in undifferentiated embryonic stem cells and embryonic germ cells (1). A network of key factors that includes Oct-4, Nanog, and Sox2 is necessary for the maintenance of pluripotent potential, and downregulation of Oct-4 has been shown to trigger cell differentiation (2,3). Research studies have demonstrated that Oct-4 is a useful germ cell tumor marker (4). Oct-4 exists as two splice variants, Oct-4A and Oct-4B (5). Recent studies have suggested that the Oct-4A isoform has the ability to confer and sustain pluripotency, while Oct-4B may exist in some somatic, non-pluripotent cells (6,7).

  1. Looijenga, L.H. et al. (2003) Cancer Res 63, 2244-50.
  2. Pesce, M. and Schöler, H.R. (2001) Stem Cells 19, 271-278.
  3. Pan, G. and Thomson, J.A. (2007) Cell Res 17, 42-9.
  4. Cheng, L. et al. (2007) J Pathol 211, 1-9.
  5. Takeda, J. et al. (1992) Nucleic Acids Res 20, 4613-20.
  6. Cauffman, G. et al. (2006) Stem Cells 24, 2685-91.
  7. Lee, J. et al. (2006) J Biol Chem 281, 33554-65.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP C&R: CUT&RUN

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 3
#83932

Oct-4A (D6C8T) Rabbit mAb

Western Blotting Image 1: Oct-4A (D6C8T) Rabbit mAb Expand Image
Western blot analysis of extracts from F9 and NIH/3T3 cells using Oct-4A (D6C8T) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). As expected, NIH/3T3 cells do not express Oct-4A.
Immunoprecipitation Image 1: Oct-4A (D6C8T) Rabbit mAb Expand Image
Immunoprecipitation of Oct-4A from F9 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Oct-4A (D6C8T) Rabbit mAb. Western blot analysis was performed using Oct-4A (D6C8T) Rabbit mAb.
Immunofluorescence Image 1: Oct-4A (D6C8T) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of F9 (left) and NIH/3T3 (right) cells using Oct-4A (D6C8T) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red).
Flow Cytometry Image 1: Oct-4A (D6C8T) Rabbit mAb Expand Image
Flow cytometric analysis of NIH/3T3 cells (blue) and F9 cells (green) using Oct-4A (D6C8T) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin Immunoprecipitation Image 1: Oct-4A (D6C8T) Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from mES cells and either Oct-4A (D6C8T) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP® Mouse Oct-4 Promoter Primers #4653, SimpleChIP® Mouse XIST Intron 1 Primers #4659, and SimpleChIP® Mouse RPL30 Intron 2 Primers #7015. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
CUT and RUN Image 1: Oct-4A (D6C8T) Rabbit mAb Expand Image
CUT&RUN was performed with mES cells and Oct-4A (D6C8T) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figure shows binding across Emc10 gene, a known target gene of Oct-4A (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 2: Oct-4A (D6C8T) Rabbit mAb Expand Image
CUT&RUN was performed with mES cells and Oct-4A (D6C8T) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 7 (upper), including Emc10 (lower), a known target gene of Oct-4A (see additional figure containing CUT&RUN-qPCR data).
CUT and RUN Image 3: Oct-4A (D6C8T) Rabbit mAb Expand Image
CUT&RUN was performed with mES cells and either Oct-4A (D6C8T) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using mouse Emc10 promoter primers, mouse Nes promoter primers, and mouse Frmd3 promoter primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.