Podocalyxin-Like (PODXL) antibody

Antigen: Podocalyxin-Like (PODXL)

Synonyms: PC, PCLP, Gp200, MGC138240, Ly102, Pclp1, PCLP-1, AW121214, podocalyxin, PODXL, podxl, fc18d02, wu:fc18d02

Clonality: Monoclonal (TRA-1-60)

Host: Mouse

Reactivity: Human

Conjugate: Un-conjugated

Application: Western Blotting (WB), BioImaging (BI), Flow Cytometry (FACS), Immunocytochemistry (ICC), Immunoprecipitation (IP), Radioimmunoassay (RIA)

Catalog no.: ABIN967659

Additional Information

Alternative name: TRA-1-60

Immunogen: Human Embryonal Carcinoma Cell Line

Cross-Reactivity: Rhesus Monkey

Format: Liquid

Isotype: IgM

Clone: TRA-1-60

Description: The TRA-1-60 monoclonal antibody reacts with the neuraminidase-resistant form of a pluripotent-stem-cell-specific epitope on a high-molecular-weight transmembrane glycoprotein. The TRA-1-60 antigen is a sialylated epitope on the same keratan sulfate core molecule, podocalyxin, as 4 other distinct antigens on tumor-derived cell lines, TRA-1-81, GCTM2, K4, and K21. The expression of TRA-1-60 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. TRA-1-60 antigen is released into the serum of patients bearing testicular tumors containing EC cells. As human EC and ES cells undergo differentiation, expression of TRA-1-60 antigen is lost. Expression of TRA-1-60 antigen has also been observed on a rhesus monkey ES cell line (Thomson et al, 1995).
Synonyms: TRA-1-60(R)

Characteristics: 1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. 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.
3. Please refer to us for technical protocols.
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.

Molecular Weight: Multiple, >200 kDa

Comments:

Related Products: ABIN967389

Application Details

Application Notes: Bioimaging:
1. Seed the cells in appropriate culture medium at an appropriate cell density in an 96-well Imaging Plate , and culture overnight to 48 hours.
2. Remove the culture medium from the wells, wash the wells twice with 100 myl of 1× PBS, and fix the cells by adding 100 µl of fresh 3.7% Formaldehyde in PBS or fixation buffer 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 myl 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 myl 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 myl of 1× PBS.
8. Remove the PBS, and counter-stain the nuclei by adding 100 ml of a 2 mg/ml solution of Hoechst 33342 in 1× PBS to each well at least 15 minutes before imaging.
9. View and analyze the cells on an appropriate imaging instrument.

Concentration: 0.5 mg/ml

Purity: Purified

Purification: Purified from tissue culture supernatant or ascites by affinity chromatography.

Buffer: Aqueous buffered solution.

Preservative: 0.09% Sodium azide.

Storage: Store undiluted at 4°C.

Restrictions: For Research Use only

Images

Western Blot analysis of TRA-1-60 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-1-60 Antigen monoclonal antibody 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-1-60 epitope are identified above 200 kDa in the human ES cells. Expression of the TRA-1-60 epitope has not been reported in mouse ES 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.

Immunofluorescent staining of human ES cell line. The H9 cell line (WiCell, Madison, WI) was cultured, fixed, and stained with purified mouse anti-human TRA-1-60 Antigen monoclonal antibody (clone TRA-1-60) (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-1-60 mAb, and the right image shows TRA-1-60 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. If permeabilization is required for staining additional markers, we recommend the use of cold 90% methanol or BD™ Phosflow Perm Buffer III.

Immunofluorescent staining of human ES cell line

Publications

Product: Andrews, Banting, Damjanov et al.: "Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells." in: Hybridoma, Vol. 3, Issue 4, pp. 347-61, 1985 (PubMed).

Thomson, Kalishman, Golos et al.: "Isolation of a primate embryonic stem cell line." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 92, Issue 17, pp. 7844-8, 1995 (PubMed).

Thomson, Itskovitz-Eldor, Shapiro et al.: "Embryonic stem cell lines derived from human blastocysts." in: Science (New York, N.Y.), Vol. 282, Issue 5391, pp. 1145-7, 1998 (PubMed).

Badcock, Pigott, Goepel et al.: "The human embryonal carcinoma marker antigen TRA-1-60 is a sialylated keratan sulfate proteoglycan." in: Cancer research, Vol. 59, Issue 18, pp. 4715-9, 1999 (PubMed).

Draper, Pigott, Thomson et al.: "Surface antigens of human embryonic stem cells: changes upon differentiation in culture." in: Journal of anatomy, Vol. 200, Issue Pt 3, pp. 249-58, 2002 (PubMed).

Henderson, Draper, Baillie et al.: "Preimplantation human embryos and embryonic stem cells show comparable expression of stage-specific embryonic antigens." in: Stem cells (Dayton, Ohio), Vol. 20, Issue 4, pp. 329-37, 2002 (PubMed).

Schopperle, DeWolf: "The TRA-1-60 and TRA-1-81 human pluripotent stem cell markers are expressed on podocalyxin in embryonal carcinoma." in: Stem cells (Dayton, Ohio), Vol. 25, Issue 3, pp. 723-30, 2007 (PubMed).