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Recombinant SUMO-GFP Protease Substrate Protein, CF

Catalog #: 11698-SO Datasheet / COA / SDS
Catalog # Availability Size / Price Qty
11698-SO-100

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ULP1 and SENP1 cleavage of Recombinant SUMO-GFP Protease Substrate Protein.
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Recombinant SUMO-GFP Protease Substrate Protein, CF Summary

  • R&D Systems E. coli-derived Recombinant SUMO-GFP Protease Substrate Protein (11698-SO)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Product Specifications

Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its cleavage by SUMO Protease ULP1.
>60% of 1 μg can be cleaved by 1 ng of SUMO Protease ULP1, as measured under the described conditions.
Source
E. coli-derived crystal jellyfish SUMO-GFP Protease Substrate protein
Ser2-Lys238, with an N-terminal Met, 6-His tag, and Sumo tag
Accession #
AAA58246
N-terminal Sequence
Analysis
Met
Predicted Molecular Mass
39 kDa
SDS-PAGE
42-47 kDa, under reducing conditions.

Product Datasheets

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11698-SO

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.

11698-SO

Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
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: 50 mM Tris, 5 mM DTT, pH 8.0 
  • Recombinant SUMO-GFP Protease Substrate (rSUMO-GFP) (Catalog # 11698-SO) 
  • Recombinant Yeast SUMO Protease ULP1 His-tag (SUMO Protease ULP1) (Catalog # 11697-SO) 
  • 15% SDS-PAGE gel
  • Reducing Sample Buffer
  • Gel Staining Reagent
  1. Dilute rSUMO-GFP to 200 µg/mL in Assay Buffer.
  2. Dilute SUMO Protease ULP1 to 0.2 µg/mL in Assay buffer.
  3. Combine 20 µL of 0.2 µg/mL SUMO Protease ULP1and 20 µL of 200 µg/mL rSUMO-GFP. Prepare a Negative Control by combining 20 µL of 200 µg/mL rSUMO-GFP and 20 µL Assay Buffer.
  4. Incubate reaction mixtures and control at room temperature for 2 hours.
  5. Stop the reactions by combining equal volumes of reactions (including control) and Reducing SDS-PAGE Sample Buffer. Heat all samples at 95 °C for 3 minutes.
  6. Load 20 µL of each stopped reaction per lane on a 15% SDS-PAGE gel and perform electrophoresis.
  7. Stain gel and analyze the % cleavage of rSUMO-GFP using densitometry.
Per Well:
  • rSUMO-GFP: 1.0 µg
  • SUMO Protease ULP1: 1.0 ng 

Scientific Data

Enzyme Activity View Larger

Recombinant SUMO-GFP Protease Substrate (Catalog # 11698-SO) can be efficiently cleaved by SUMO Protease ULP1 (11697-SO) and Recombinant Human His6-SENP1 Catalytic Domain Protein (E-700).

SDS-PAGE View Larger

2 μg/lane of Recombinant SUMO-GFP Protease Substrate Protein (Catalog # 11698-SO) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 42-47 kDa, under reducing conditions.

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: SUMO-GFP Protease Substrate

SUMO-GFP Protease Substrate is a recombinant protein that contains SUMO fused to GFP that can be used as a positive control in an SDS-PAGE gel-shift assay to detect cleavage activity by Recombinant SUMO Protease ULP1 (Catalog # 11697-SO) and Recombinant Human His6-SENP1 Catalytic Domain Protein
(Catalog # E-700). SUMO is a member of the small ubiquitin-like modifier (SUMO) family. In the cell, the SUMO protein is modified and covalently conjugated to other proteins in the cell via an isopeptide bond formed between its carboxyl-terminal glycine residues and the lysine of the protein substrate in a similar parallel mechanism of ubiquitination that regulates cellular processes such as DNA replication and repair, nuclear transport, signal transduction, and regulation of transcription (1-3). Use of SUMO fusions as a technology has been used to enhance protein expression, solubility, and purification in prokaryotes for a diverse population of protein targets and unlike other common fusion partners SUMO can serve as a chaperone for correct folding (4).  SUMO is cleaved at its c-terminus to a mature form after a glycine-glycine pair by a family of sentrin/SUMO-specific proteases (SENPs) in humans and ubiquitin-like protein specific proteases (Ulps) in yeast to regulate the reversible process of sumoylation (3). In a fusion application, the cleavage of SUMO allows for successful recombinant protein production with a native N-terminal sequence providing a distinct advantage over other fusion cleavage models used in recombinant protein production in both academic research and industrial biotechnology (4‑10).

References
  1. Li, S.J and M. Hochstrasser (1999) Nature 398:246.
  2. Li, S.J and M. Hochstrasser (2000) Mol. Cell Biol. 20:2367.
  3. Mukhopadhyay, D. and M. Dasso. (2007) Trends Biochem. Sci. 32:286.
  4. Malakhov, M.P. et al. (2004) J. Struct. Funct. Genomics 5:75.  
  5. Marblestone, J.G. et al. (2006) Protein Sci. 15:182.
  6. Baker, R.T. (1996) Curr. Opin. Biotechnol. 7:541.
  7. Jonasson, P. et al. (2002) Biotechnol. Appl. Biochem. 35:91.
  8. Butt, T.R. et al. (2005) Protein Expr. Purif. 43:1.
  9. Peroutka, R.J. et al. (2011) Methods Mol. Biol. 705:15.
  10. Wang, Z. et al. (2010) Protein Expr. Purif. 73:203.

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