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Purified Mouse anti-Human TRA-1-81 Antigen
Purified Mouse anti-Human TRA-1-81 Antigen

Left Panel: Western Blot analysis of TRA-1-81 in mouse and human ES cell lines. Lysates from ES-E14TG2a mouse ES cells (ATCC CRL-1821, left blot) and H9 human ES cells* (WiCell, Madison, WI, right blot) were probed with Purified Mouse anti-Human TRA-1-81 Antigen (Cat. No. 560072) at titrations of 2.0 (lanes 1), 1.0 (lanes 2), and 0.5 µg/ml (lanes 3).  High-molecular-weight molecules bearing the TRA-1-81 epitope are identified above 200 kDa in the human ES cells.  Expression of the TRA-1-81 epitope has not been reported in mouse ES cells.  

Center Panels: Immunofluorescent staining of human ES cell line.  The H9 cell line was cultured, fixed, and stained with Purified Mouse anti-Human TRA-1-81 Antigen monoclonal antibody (pseudo-colored green) according to the Recommended Assay Procedure.  The second-step reagent was Alexa Fluor® 647 goat anti-mouse Ig (Invitrogen).  The left image shows the plasma membrane staining by the TRA-1-81 mAb, and the right image shows TRA-1-81 with counter-staining of the nuclei by Hoechst 33342 (pseudo-colored blue).  The images were captured on a BD Pathway™ 435 Cell Analyzer using a 10X objective and merged using BD Attovision™ software.

Right Panel: Flow cytometric analysis of TRA-1-81 antigen expression on NCCIT (human pluripotent embryonal carcinoma) cell line. NCCIT cells (ATCC CRL-20730) were stained with either Purified Mouse anti-Human TRA-1-81 Antigen (solid line histogram) or Purified Mouse IgM, κ Isotype Control (Cat. No. 555581; dashed line histogram), followed by FITC Goat Anti-Mouse Ig (Cat. No. 554001). Fluorescent histograms were derived from gated events with the side and forward light-scattering characteristics of viable cells.

*The H9 cells were cultured on a mitomycin C-treated mouse embryonic fibroblast feeder layer [MEF (CF-1), ATCC SCRC-1040] that maintains the undifferentiated state of the ES cells.  The lysate was made from a mixture of the 2 cell types, the majority of which were H9 cells.

Left Panel: Western Blot analysis of TRA-1-81 in mouse and human ES cell lines. Lysates from ES-E14TG2a mouse ES cells (ATCC CRL-1821, left blot) and H9 human ES cells* (WiCell, Madison, WI, right blot) were probed with Purified Mouse anti-Human TRA-1-81 Antigen (Cat. No. 560072) at titrations of 2.0 (lanes 1), 1.0 (lanes 2), and 0.5 µg/ml (lanes 3).  High-molecular-weight molecules bearing the TRA-1-81 epitope are identified above 200 kDa in the human ES cells.  Expression of the TRA-1-81 epitope has not been reported in mouse ES cells.  

Center Panels: Immunofluorescent staining of human ES cell line.  The H9 cell line was cultured, fixed, and stained with Purified Mouse anti-Human TRA-1-81 Antigen monoclonal antibody (pseudo-colored green) according to the Recommended Assay Procedure.  The second-step reagent was Alexa Fluor® 647 goat anti-mouse Ig (Invitrogen).  The left image shows the plasma membrane staining by the TRA-1-81 mAb, and the right image shows TRA-1-81 with counter-staining of the nuclei by Hoechst 33342 (pseudo-colored blue).  The images were captured on a BD Pathway™ 435 Cell Analyzer using a 10X objective and merged using BD Attovision™ software.

Right Panel: Flow cytometric analysis of TRA-1-81 antigen expression on NCCIT (human pluripotent embryonal carcinoma) cell line. NCCIT cells (ATCC CRL-20730) were stained with either Purified Mouse anti-Human TRA-1-81 Antigen (solid line histogram) or Purified Mouse IgM, κ Isotype Control (Cat. No. 555581; dashed line histogram), followed by FITC Goat Anti-Mouse Ig (Cat. No. 554001). Fluorescent histograms were derived from gated events with the side and forward light-scattering characteristics of viable cells.

*The H9 cells were cultured on a mitomycin C-treated mouse embryonic fibroblast feeder layer [MEF (CF-1), ATCC SCRC-1040] that maintains the undifferentiated state of the ES cells.  The lysate was made from a mixture of the 2 cell types, the majority of which were H9 cells.

Product Details
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BD Pharmingen™
Human (QC Testing), Rhesus (Reported)
Mouse BALB/c IgM, κ
Human Embryonal Carcinoma Cell Line
Flow cytometry (Routinely Tested), Bioimaging, Western blot (Tested During Development), Immunoprecipitation, Radioimmunoassay (Reported)
multiple, >200 kDa
0.5 mg/ml
AB_1645605
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO


Preparation And Storage

Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

Recommended Assay Procedures

Bioimaging

1.        Seed the cells in appropriate culture medium at an appropriate cell density in a 96-well Imaging Plate, and

        culture overnight to 48 hours.

2.        Remove the culture medium from the wells, wash the wells twice with 100 μl of 1× PBS, and fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or BD Cytofix™ fixation buffer (Cat. No. 554655) to each well and incubating for 10 minutes at room temperature (RT).

3.        Remove the fixative from the wells, and wash the wells twice with 100 μl of 1× PBS.

4.        Dilute the antibody in 1× PBS, and stain the cells by adding 50 µl of the diluted antibody to each well and incubating for 1 hour at RT.

5.        Remove the diluted antibody, and wash the wells three times with 100 μl of 1× PBS.

6.        Remove the PBS, dilute the second-step reagent in 1× PBS, and stain the cells by adding 50 µl of the diluted second-step reagent to each well and incubating for 1 hour at RT.

7.        Remove the diluted second-step reagent, and wash the wells twice with 100 μl of 1× PBS.

8.        Remove the PBS, and counter-stain the nuclei by adding 100 μl of a 2 μg/ml solution of Hoechst 33342 (eg, Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.

9.        View and analyze the cells on an appropriate imaging instrument.

For more detailed information on Bioimaging and Western blot applications, please refer to "Cellular Imaging" and "Cell Biology (WB, IP, IHC, IF)" at our website: http://www.bdbiosciences.com/us/s/resources

Product Notices

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. An isotype control should be used at the same concentration as the antibody of interest.
  3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  4. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
  5. Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
  6. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
  7. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
560072 Rev. 4
Antibody Details
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TRA-1-81

The TRA-1-81 monoclonal antibody reacts with a pluripotent stem cell-specific epitope on a high molecular weight transmembrane glycoprotein.  The TRA-1-81 antigen is an epitope on the same keratan sulfate core molecule, podocalyxin, as 4 other distinct antigens on tumor-derived cell lines, TRA-1-60, GCTM2, K4, and K21.  The expression of TRA-1-81 antigen is stage-specific and can be used to characterize embryonic cells and monitor their differentiation.  The antigen is found on teratocarcinoma (embryonal carcinoma or EC), embryonic inner cell mass (but not morula or trophoblast), and embryonic stem (ES) cells.  As human EC and ES cells undergo differentiation, expression of TRA-1-81 antigen is lost.

  

560072 Rev. 4
Format Details
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
560072 Rev.4
Citations & References
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Development References (7)

  1. Andrews PW, Banting G, Damanov I, Arnaud D, Avner P. Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells. Hybridoma. 1984; 3(4):347-361. (Immunogen: Immunofluorescence, Immunoprecipitation, Radioimmunoassay). View Reference
  2. Badcock G, Pigott C, Goepel J, Andrews PW. The human embryonal carcinoma marker antigen TRA-1-60 is a sialylated keratan sulfate proteoglycan. Cancer Res. 1999; 59:4715-4719. (Clone-specific: Immunoprecipitation, Western blot). View Reference
  3. Draper JS, Pigott C, Thomson JA, Andrews PW. Surface antigens of human embryonic stem cells: changes upon differentiation in culture. J Anat. 2002; 200:249-258. (Clone-specific: Flow cytometry). View Reference
  4. Henderson JK, Draper JS, Baillie HS, et al. Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens. Stem Cells. 2002; 20:329-337. (Clone-specific: Flow cytometry, Immunofluorescence). View Reference
  5. Schopperle WM, DeWolf WC. The TRA-1-60 and TRA-1-81 human pluripotent stem cell markers are expressed on podocalyxin in embryonal carcinoma. Stem Cells. 2007; 25:723-730. (Clone-specific: Immunofluorescence, Western blot). View Reference
  6. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998; 282:1145-1147. (Clone-specific: Immunocytochemistry (cytospins)). View Reference
  7. Thomson JA, Kalishman J, Golos TG, et al. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A. 1995; 92:7844-7848. (Clone-specific: Immunocytochemistry (cytospins)). View Reference
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560072 Rev. 4

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Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described


Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims.  Comparisons are not made against non-BD technologies, unless otherwise noted.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

Refer to manufacturer's instructions for use and related User Manuals and Technical Data Sheets before using this product as described.

Comparisons, where applicable, are made against older BD technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.