Alexa Fluor® 647 Mouse anti-Human TRA-1-81 Antigen
Clone TRA-1-81 (RUO)
- Brand BD Pharmingen™
- Vol. Per Test 5 µl
- Isotype Mouse BALB/c IgM, κ
- Reactivity Human (QC Testing) Rhesus (Reported)
Bioimaging (Routinely Tested)
- Immunogen Human Embryonal Carcinoma Cell Line
- Storage Buffer Aqueous buffered solution containing BSA, protein stabilizer, and ≤0.09% sodium azide.
- Regulatory Status RUO
Regulatory Status Legend
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.
Alexa Fluor® 647 conjugates are highly photostable and remain fluorescent over a broad pH range. The excitation and emission maxima are nearly identical to those of APC. However, APC tends to be brighter while Alexa Fluor® 647 is more optimal for intracellular applications. This fluorochrome exhibits uncommon photostability, making it an ideal choice for use in fluorescence microscopy. Due to nearly identical excitation and emission properties but different spillover characteristics, APC and Alexa Fluor® 647 cannot be used simultaneously.
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Preparation and Storage
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
The antibody was conjugated to Alexa Fluor® 647 under optimum conditions, and unreacted Alexa Fluor® 647 was removed.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze.
- Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.
- This reagent has been pre-diluted for use at the recommended Volume per Test when following the Recommended Assay Procedure. A Test is typically ~10,000 cells cultured in a well of a 96-well imaging plate.
- The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
1. Seed the cells in appropriate culture medium at an appropriate cell density in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219), 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 conjugate to each well and incubating for 1 hour at RT.
5. Remove the diluted antibody, and wash the wells twice with 100 μl of 1× PBS.
6. 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.
7. View and analyze the cells on an appropriate imaging instrument.