Recombinant Human TPST2 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human TPST2 Protein, CF Summary

• Details of Functionality: 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: No N-terminal sequence was revealed by sequencing. The Gln residue maybe blocked. Protein identity confirmed by MS analysis of tryptic fragments.
• Protein/Peptide Type: Recombinant Enzymes
• Gene: TPST2
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 40 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 40-50 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Assay Procedure:
  • 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 PAP^35S)

Per Reaction:

• PAPS: 1000 pmol (pmol of PAP³⁵S in the assay is negligible)
• rhTPST2: 0.25, 0.125, 0.0625 and 0.0312 μg

FAQs for Tyrosylprotein Sulfotransferase 2/TPST2 (6236-ST). (Showing 1 - 1 of 1 FAQ).

1. What is the lower limit of detection for TPST2 in Western blot?
   • We generally do not test the limits of detection for our antibodies as this time. I apologize for the inconvenience.

Bioinformatics

• Gene Symbol: TPST2
• Uniprot:
  • O60704()