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Recombinant Human TPST2 Protein, CF

Catalog #: 6236-ST Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
6236-ST-020
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Recombinant Human TPST2 Protein, CF Summary

Product Specifications

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to transfer sulfate from PAPS to PSGL-1 peptide (Gln-Ala-Thr-Glu-Tyr-Glu-Tyr-Leu-Asp-Tyr-Asp-Phe-Leu-Pro-Glu-Thr) The specific activity is >20 pmol/min/μg, as measured under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived human Tyrosylprotein Sulfotransferase 2/TPST2 protein
Gln26-Ser377, with a C-terminal 6-His tag
Accession #
O60704
N-terminal Sequence
Analysis
No N-terminal sequence was revealed by sequencing. The Gln residue maybe blocked. Protein identity confirmed by MS analysis of tryptic fragments.
Predicted Molecular Mass
40 kDa
SDS-PAGE
40-50 kDa, reducing conditions

Product Datasheets

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6236-ST

Product Datasheet
COA
SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

6236-ST

Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • Assay Buffer: 25 mM MES, 0.5% (v/v) Triton® X-100, 2.5 mM MgCl2, 2.5 mM MnCl2, 1.25 mM CaCl2, 0.75 mg/mL BSA, pH 7.0
  • Gel Running Buffer: 40 mM Tris, 1 mM EDTA, adjust to pH 8.0 with acetic acid
  • Recombinant Human Tyrosylprotein Sulfotransferase 2/TPST2 (rhTPST2) (Catalog # 6236-ST)
  • 8% SDS-PAGE (approximately 15 cm x 20 cm, 20 lanes per gel)
  • PSGL-1 peptide (QATEYEYLDYDFLPET), custom synthesized, 5 mM stock in 25 mM MES pH 7.0
  • PAPS (Sigma, Catalog # A1651), 1 mM stock solution in 5% ethanol, 95% deionized water
  • PAP35S (prepared in-house) (12-14), ~1 μM in 5% ethanol, 95% deionized water
  • Gel loading buffer: 0.15 M Tris, 20.8 mM SDS, 1.15 M Glycine, 174 µM Bromophenol Blue, 30% Glycerol
  • Blotting paper (Fisher Scientific, Catalog # 05-714-4)
  • Gel dryer
  • Glogos® II autorad markers (Stratagene, Catalog # 420202) or equiv.
  • Blue sensitive medical X-ray film
  • X-ray film cassette
  • Film developer (Konica SRX-101A Medical Film Processor) or equiv.
  • Liquid scintillation counter (Beckman Coulter, Model # LS5000TD) or equiv.
  • Liquid scintillation fluid (Beckman Coulter, Catalog # 141349) or equiv.
  1. Dilute rhTPST2 to 16.67, 8.33, 4.16 and 2.08 µg/mL in Assay Buffer.
  2. Combine 10 µL PAPS, 10 µL PAP35S, 35 µL PSGL-1 peptide, and 95 µL deionized water (rxn mix sufficient for ~9 rxns).
  3. Combine 15 µL enzyme at each dil. with 15 µL rxn mix. Include a control containing 15 µL Assay Buffer and 15 µL rxn mix.
  4. Incubate at 37 °C for 20 min.
  5. Add 15 µL gel loading buffer to each rxn. Mix.
  6. Load 30 µL of each rxn per lane on a gel. Leave empty lanes between samples. Run at 200 V for 25 min.
  7. Transfer gel onto blotting paper and dry with gel dryer for 1 hr or until fully dry.
  8. Affix two autorad markers to the blotting paper next to the dried gel.
  9. In a darkroom expose dried gel to X-ray film by enclosing overnight in film cassette.
  10. After developing, use autorad markers to align X-ray film with gel.
  11. Determine where labeled product (slowest), unreacted PAP35S (intermediate), and free sulfate (fastest) migrated on the gel. For the control, identify the empty region where the product would have migrated if any had been produced.
  12. Cut out regions containing product and unreacted PAP35S.
  13. Place each region into a separate liquid scintillation vial. Add 5 mL liquid scintillation fluid to each vial.
  14. Use a liquid scintillation counter to count the amount of 35S in each vial.
  15. Calculate Activity for each rxn using the following equation. Plot Activity vs. μg per reaction, fit to linear regression. The slope of the fit equals the Specific Activity (pmol/min/μg):

     Activity (pmol/min) =

 S (pmol) x Ci (counts)
Ct (counts) x time (min)
S = applied donor substrate Ci = incorporated radioisotope (product) Ct = total applied radioisotope (product plus unreacted PAP35S)
Per Reaction:
  • PAPS: 1000 pmol (pmol of PAP35S in the assay is negligible)
  • rhTPST2: 0.25, 0.125, 0.0625 and 0.0312 μg
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Tyrosylprotein Sulfotransferase 2/TPST2

Tyrosine O-sulfation is a posttranslational modification found in all multicellular organisms (1, 2, 3). This reaction is mediated by tyrosylprotein sulfotransferase (TPST), a Golgi enzyme that transfers sulfate from 3'-phosphoadenosine 5'‑phosphosulfate (PAPS) to tyrosine residues contained in polypeptides with acidic motifs to form a tyrosine O-sulfate ester. More than 60 proteins have been identified to be tyrosine sulfated. The function of this modification have been identified in some cases. For example, sulfation of tyrosine residues in the leukocyte adhesion molecule P‑selectin glycoprotein ligand 1 (PSGL-1) is required for binding to P‑selectin on activated endothelium (4, 5), and tyrosine sulfation of chemokine receptors CCR5 and CXCR4 has been reported to facilitate HIV-1 entry of target cells (6, 7). Two TPSTs are found in the human genome. Compared to TPST1, TPST2 has similar tissue distribution and overlapping substrate specificity (8, 9). In contrast to
Tpst1-/- males with normal fertility in mice, Tpst2-/- males are infertile due to severe defects in sperm motility (10). The enzymatic activity was assayed using an SDS-PAGE based method (11).

References
  1. Kehoe, J.W. and Bertozzi, C.R. (2000) Chemistry & Biology 7:R57.
  2. Huttner, W.B. (1988) Annu. Rev. Physiol. 50:363.
  3. Ouyang, Y. et al. (1998) Proc. Natl. Acad. Sci. USA 95:2896.
  4. Danan, L.M. et al. (2008) J. Am. Soc. Mass. Spectrom. 19:1459.
  5. Somers, W.S. et al. (2000) Cell 103:467.
  6. Choe, H. et al. (2003) Cell 114:161.
  7. Seibert, C. et al. (2008) Biochemistry 47:11251.
  8. Ouyang, Y.B. and Moore, K.L. (1998) J. Biol. Chem. 273:24770.
  9. Jen, C.H. (2009) Biochemistry 48:5332.
  10. Borghei, A. et al. J. Biol. Chem. 281:9423.
  11. Wu, Z.L. et al. (2010) BMC Biotechnol. 10:11.
  12. Robbins, P.W. (1962) Methods in Enzymology, Vol. V, Academic Press, Inc., New York, 964.
  13. MacRae, I.J. et al. (2000) Biochemistry 39:1613.
  14. Wu, Z.L. et al. (2002) FASEB J. 16:539.
Entrez Gene IDs
8459 (Human)
Alternate Names
EC 2.8.2.20; TPST2; TPST-2; tyrosylprotein phosphotransferase 2; Tyrosylprotein Sulfotransferase 2; tyrosylprotein sulfotransferase 2protein-tyrosine sulfotransferase 2; tyrosylprotein sulfotransferase-2

Product Specific Notices

Coomassie is a registered trademark of Imperial Chemical Industries Ltd. Triton is a registered trademark of Union Carbide Corp. Glogos is a registered trademark of Stratagene.

FAQs

No product specific FAQs exist for this product, however you may

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Sulfotransferase Assays and Substrates

3'-Phosphoadenosine-5'-phosphosulfate

ES019
3 Citations

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