Recombinant Human TPST2 Protein, CF Summary
Gln26-Ser377, with a C-terminal 6-His tag
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.
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.
|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.
- 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.
- Dilute rhTPST2 to 16.67, 8.33, 4.16 and 2.08 µg/mL in Assay Buffer.
- Combine 10 µL PAPS, 10 µL PAP35S, 35 µL PSGL-1 peptide, and 95 µL deionized water (rxn mix sufficient for ~9 rxns).
- 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.
- Incubate at 37 °C for 20 min.
- Add 15 µL gel loading buffer to each rxn. Mix.
- Load 30 µL of each rxn per lane on a gel. Leave empty lanes between samples. Run at 200 V for 25 min.
- Transfer gel onto blotting paper and dry with gel dryer for 1 hr or until fully dry.
- Affix two autorad markers to the blotting paper next to the dried gel.
- In a darkroom expose dried gel to X-ray film by enclosing overnight in film cassette.
- After developing, use autorad markers to align X-ray film with gel.
- 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.
- Cut out regions containing product and unreacted PAP35S.
- Place each region into a separate liquid scintillation vial. Add 5 mL liquid scintillation fluid to each vial.
- Use a liquid scintillation counter to count the amount of 35S in each vial.
- 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)|
- PAPS: 1000 pmol (pmol of PAP35S in the assay is negligible)
- rhTPST2: 0.25, 0.125, 0.0625 and 0.0312 μg
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).
- Kehoe, J.W. and Bertozzi, C.R. (2000) Chemistry & Biology 7:R57.
- Huttner, W.B. (1988) Annu. Rev. Physiol. 50:363.
- Ouyang, Y. et al. (1998) Proc. Natl. Acad. Sci. USA 95:2896.
- Danan, L.M. et al. (2008) J. Am. Soc. Mass. Spectrom. 19:1459.
- Somers, W.S. et al. (2000) Cell 103:467.
- Choe, H. et al. (2003) Cell 114:161.
- Seibert, C. et al. (2008) Biochemistry 47:11251.
- Ouyang, Y.B. and Moore, K.L. (1998) J. Biol. Chem. 273:24770.
- Jen, C.H. (2009) Biochemistry 48:5332.
- Borghei, A. et al. J. Biol. Chem. 281:9423.
- Wu, Z.L. et al. (2010) BMC Biotechnol. 10:11.
- Robbins, P.W. (1962) Methods in Enzymology, Vol. V, Academic Press, Inc., New York, 964.
- MacRae, I.J. et al. (2000) Biochemistry 39:1613.
- Wu, Z.L. et al. (2002) FASEB J. 16:539.
Product Specific NoticesCoomassie 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.
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Sulfotransferase Assays and Substrates
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