Cell Signaling Technology

Product Pathways - Stem Cell and Lineage Markers

StemLight™ Pluripotency Antibody Kit #9656

Kit Includes Quantity Applications Dilution Isotype
Oct-4A (C30A3) Rabbit mAb 100 tests IF-IC 1:200 Rabbit IgG
Sox2 (D6D9) XP® Rabbit mAb 100 tests IF-IC 1:200 Rabbit IgG
Nanog Antibody 100 tests IF-IC 1:200 Rabbit IgG
SSEA4 (MC813) Mouse mAb 100 tests IF-IC 1:200 Mouse IgG3
TRA-1-60(S) (TRA-1-60(S)) Mouse mAb 100 tests IF-IC 1:200 Mouse IgM
TRA-1-81 (TRA-1-81) Mouse mAb 100 tests IF-IC 1:200 Mouse IgM

Applications Key:  IF-IC=Immunofluorescence (Immunocytochemistry)

Specificity / Sensitivity

Each antibody in the StemLight™ Pluripotency Antibody Kit detects endogenous levels of their respective human pluripotency marker proteins.

IF-IC

IF-IC

Projected confocal z-stack of human iPS cells using TRA-1-60(S) (TRA-1-60(S)) Mouse mAb (green, upper right), TRA-1-81 (TRA-1-81) Mouse mAb (green, upper middle), SSEA4 (MC813) Mouse mAb (green, upper left), Oct-4A (C30A3) Rabbit mAb (green, lower right), Sox2 (D6D9) XP® Rabbit mAb (green, lower middle) and Nanog Antibody (green, lower left). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

IF-IC

IF-IC

Confocal immunofluorescent analysis of NTERA-2 cells, untreated (left panel) or retinoic acid-treated (10 µM all-trans RA for 5 days) (right panel), using TRA-1-60(S) (TRA-1-60(S)) Mouse mAb (green, upper), TRA-1-81 (TRA-1-81) Mouse mAb (green, middle) and SSEA4 (MC813) Mouse mAb (green, lower). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Note the loss of pluripotency markers (green) as cells differentiate along the neuronal lineage with retinoic acid treatment.

IF-IC

IF-IC

Confocal immunofluorescent analysis of NTERA-2 cells, untreated (left panel) or retinoic acid-treated (10 µM all-trans RA for 14 days) (right panel), using Oct-4A (C30A3) Rabbit mAb (green, upper), Sox2 (D6D9) XP® Rabbit mAb (green, middle) and Nanog Antibody (green, lower). Actin filaments have been labeled with DY-554 phalloidin (red). Note the loss of pluripotency markers (green) as cells differentiate along the neuronal lineage with retinoic acid treatment.


IF-IC

IF-IC

Confocal immunofluorescent analysis of NTERA-2 cells, untreated (left panel) or retinoic acid-treated (10 µM all-trans RA for 5 days) (right panel), using Neurofilament-L (C28E10) Rabbit mAb #2837 (green, upper), and β3-Tubulin (TU-20) Mouse mAb #4466 (green, lower). Actin filaments have been labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Note the appearance of neuronal markers and structures as cells differentiate along the neuronal lineage with retinoic acid treatment.

Source / Purification

Nanog Antibody was produced by immunizing animals with a synthetic peptide corresponding to amino acid sequence at the amino terminus of human nanog. Antibodies are purified by Protein A and peptide affinity chromatography. Oct-4A antibody is produced by immunizing animals with recombinant protein specific to the amino terminus of human Oct-4A. Sox2 antibody is produced by immunizing animals with a synthetic peptide corresponding to amino acid sequences at the amino terminus of human Sox2. SSEA4, TRA-1-81, and TRA-1-60(S) antibodies are produced by immunizing animals with human embryonal carcinoma 2102Ep cl.2A6 cells.

Background

Pluripotency is the ability of a cell to differentiate into cell types of the three germ layers, the endoderm, ectoderm and mesoderm. It is a property shared by embryonic stem cells, embryonic carcinoma and induced pluripotent cells.Oct-4, Sox2 and Nanog are key transcriptional regulators that are highly expressed in pluripotent cells (1). Together they form a transcriptional network that maintains cells in a pluripotent state (2,3). Over-expression of Oct-4 and Sox2 along with Klf4 and c- Myc can induce pluripotency in both mouse and human somatic cells, highlighting their roles as key regulators of the transcrip- tional network necessary for renewal and pluripotency (4-5). It has also been demonstrated that overexpression of Oct-4, Sox2, Nanog and Lin28 can induce pluripotency in human somatic cells (6). Upon differentiation of pluripotent cultures, expression of Oct-4, Nanog and Sox2 is downregulated.SSEA4, TRA-1-81 and TRA-1-60 antibodies recognize antigens expressed on the cell surface of all pluripotent cells. SSEA4 recognizes a glycolipid carbohydrate epitope (7). TRA-1-60(S) and TRA-1-81 antibodies recognize different proteoglycan epitopes on variants of the same protein, podocalyxin (8). These epitopes are neurominadase sensitive and resistant, respectively. Reactivity of SSEA4, TRA-1-81 and TRA-1-60 antibodies with their respective cell surface markers are lost upon differentiation of pluripotent cells, corresponding with a loss of pluripotent potential (9).

  1. Looijenga, L.H. et al. (2003) Cancer Res 63, 2244-50.
  2. Pesce, M. and Schöler, H.R. (2001) Stem Cells 19, 271-8.
  3. Pan, G. and Thomson, J.A. (2007) Cell Res 17, 42-9.
  4. Takahashi, K. and Yamanaka, S. (2006) Cell 126, 663-76.
  5. Okita, K. et al. (2007) Nature 448, 313-7.
  6. Yu, J. et al. (2007) Science 318, 1917-20.
  7. Henderson, J.K. et al. (2002) Stem Cells 20, 329-37.
  8. Draper, J.S. et al. (2002) J Anat 200, 249-58.
  9. Schopperle, W.M. and DeWolf, W.C. (2007) Stem Cells 25, 723-30.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Protocols

Companion Products

For Research Use Only. Not For Use In Diagnostic Procedures.

Products