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SouthBayBio - Specialized in Bioassays, Enzymes and Advanced TR-FRET Technology

South Bay Bio, LLC, formed in 2016, provides expertise in the ubiquitin proteasome system (UPS) and other innovative research areas, like UPS related assay development, HTS, protein purification, bioconjugation and custom biochemistry services.

 

NEW E3 Ligase TR-FRET Assays

South Bay Bio's homogeneous Real-Time TR-FRET ubiquitin conjugation assays are simple; the format is 96 or 384 well low-volume plates (making them well suited for HTS). Using ubiquitin either labeled with Europium-Cryptate (donor) or Cyanine 5 (acceptor), for the first time ubiquitin conjugation and deconjugation can be measured homogenously in Real-Time (facilitating enzyme kinetics or endpoint if preferred), with assays commonly exhibiting Z’ 0.8 and a Signal to Noise of commonly >3000%.
Auto-ubiquitination kinetics of several human recombinant E3 ligases of significant interest, namely MDM2, MuRF1, ITCH and Parkin, along with ubiquitin conjugation on substrates such as p53 and s5a have been validated, as well as TR-FRET based deconjugation kinetics of several auto-ubiquitinated ligases using USP2cd and USP7. Coupled with the assays’ short development time (as no antibody development is required for assay optimization), the assay platform is ideally suited for a wide variety of academic and industry screening applications.

TR-FRET Kit Poster

Product Name PID Product description
ITCH E3 Ligase TR-FRET Kit SBB-KF0035 A fast and sensitive method monitoring ubiquitin conjugation onto ITCH in solution, resulting from an enzymatic ubiquitin cascade without the need of running and staining an SDS gel. The kit enables continuous TR-FRET detection of ubiquitin chain formation onto ITCH in a real-time detection setup, or in an end-point configuration if desired.
MDM2 E3 Ligase TR-FRET Kit SBB-KF0030 A fast and sensitive method monitoring ubiquitin conjugation onto GST-MDM2 in solution, resulting from an enzymatic ubiquitin cascade without the need of running and staining an SDS gel. The kit enables continuous TR-FRET detection of ubiquitin chain formation onto MDM2 in a real-time detection setup, or in an end-point configuration if desired.
Parkin E3 Ligase TR-FRET Kit SBB-KF0036 A fast and sensitive method monitoring ubiquitin conjugation onto both wild-type Parkin and a more active mutant W403A in solution, resulting from an enzymatic ubiquitin cascade without the need of running and staining an SDS gel. The kit enables continuous TR-FRET detection of ubiquitin chain formation onto Parkin in a real-time detection setup, or in an end-point configuration if desired.

Labeled Ubiquitins for TR-FRET Experiments

 

Europium-Cryptate labeled Ubiquitin

Ubiquitin (Ub) is a highly conserved protein (from yeast to mammals) that plays a major role in the ubiquitination pathway. Ubiquitination, the conjugation of ubiquitin to other proteins is essential for many cellular process primarily linked to protein degradation. The ubiquitination process involves three steps with specific groups of enzymes in an ATP-dependent manner, which are activation with ubiquitin-activating enzymes (E1s), conjugation with ubiquitin-conjugating enzymes (E2s) and ligation with ubiquitin ligases (E3s).
Good FRET pairs exhibit no overlapping spectrum between donor and acceptor, high quantum yields and red-shifted emission (e.g. Cy5) to minimize compound interference. The best donors are cryptates, comprised of rare-earth complexes (europium or terbium) with a lanthanide embedded in a macrocycle. They exhibit long-lived fluorescence, stability and robustness necessary to survive different assay conditions. Europium Cryptate-labeled Ubiquitin is ideal for measuring Ub-chain conjugation or deconjugation as a TR-FRET donor. Its ideal protein-based TR-FRET pair acceptor is Cy5-labeled Ubiquitin.

Product Name PID Product description
Ubiquitin (human) (rec.) (Europium-Cryptate) SBB-TR0014 Human ubiquitin (aa1-76) (Accession Nr. P0CG47) is site-specifically conjugated to a single Europium-Cryptate moiety. All lysines are available along with a fully functional C-terminus, making it ideal for chain formation using the E1-E2 and E3 conjugation cascade.
Ubiquitin (human) (rec.) (Cy5) SBB-TR0015 Human ubiquitin (aa1-76) (Accession Nr. P0CG47) is site-specifically conjugated to a single Cyanine 5 (Cy5) moiety. All lysines are available along with a fully functional C-terminus, making it ideal for chain formation using the E1-E2 and E3 conjugation cascade.
Ubiquitin (human) (rec.) (6-FAM) SBB-TR0016 Human ubiquitin (aa1-76) (Accession Nr. P0CG47) is site-specifically conjugated to a single fluorescein (6-FAM) moiety. All lysines are available along with a fully functional C-terminus, making it ideal for chain formation using the E1-E2 and E3 conjugation cascade.
TRF-Ubiquitin Mix (100X) (2000 reactions) SBB-TR0051-2K-1 This product is a mix of Europium-Cryptate Ubiquitin (SBB-TR0014), Cy5-Ubiquitin (SBB-TR0015), and wild-type Ubiquitin (SBB-UP0013) in a ratio optimized for TR-FRET based conjugation experiments where long polyubiquitin chains are formed. Under conditions where short ubiquitin chains are formed, a different mix may be required to optimize signal to background ratio. Enzymatic incorporation of the labeled ubiquitins into chains conjugated onto a substrate protein leads to an increase in fluorescence emission at 665nm (Em-Acceptor) and decrease at emission wavelength 620nm (Em-Donor).

Rhodamine-Labeled UB/UBL Derivatives - Protein Substrates

 

South Bay Bio's offers a growing list of Rhodamine 110-labeled protein substrates for Ubiquitin Research. These protein-based substrates have a typical working concentration range of 50-500nM. Hydrolysis of the conjugate results in fluorescence observable by excitation at 485nm and emission at 535nm.

Product Name PID Product description
Ubiquitin (human) (rec.) (Rhodamine 110) SBB-PS0001 Human ubiquitin (aa1-76) (Accession Nr. P0CG47) conjugated at the C-terminus to a quenched Rhodamine 110 dye.
ISG15 (human) (rec.) (Rhodamine 110) SBB-PS0002 Human ISG15 (aa1-157) (Accession Nr. P05161) conjugated at the C-terminus to a quenched Rhodamine 110 dye.
NEDD8 (human) (rec.) (Rhodamine 110) SBB-PS0003 Human NEDD8 (aa1-76) (Accession Nr. Q15843) conjugated at the C-terminus to a quenched Rhodamine 110 dye.
SUMO1 (human) (rec.) (Rhodamine 110) SBB-PS0028 Human SUMO1 (aa1-97) (Accession Nr. P63165) conjugated at the C-terminus to a quenched Rhodamine 110 dye.
SUMO2 (human) (rec.) (Rhodamine 110) SBB-PS0029 Human SUMO2 (aa1-93) (Accession Nr. P61956) conjugated at the C-terminus to a quenched Rhodamine 110 dye.

Active 20S Proteasome & 20S Immunoproteasome Proteins

 

Proteasome and Immunoproteasome Structure

The ubiquitin-proteasome pathway is the major proteolytic system in eukaryotic cells, where it catalyzes the selective degradation of short-lived regulatory proteins or the rapid turnover of misfolded proteins. One of the most important proteases in this pathway is the 26S proteasome, an ATP-dependent proteolytic complex, which is formed by the association of the barrel-shaped 20S proteasome (700kDa) and two 19S (700kDa) regulatory complexes. The 20S catalytic core is composed of 4 rings of 28 non-identical subunits; 2 rings are composed of 7 a-subunits and 2 rings are composed of 7 b-subunits. The 20S catalytic core is able to degrade a variety of peptide substrates and poly-ubiquitinated proteins involved in apoptosis, DNA repair, endocytosis and cell cycle control.
The immunoproteasome is structurally similar to constitutive 26S proteasome. The 20S core of immunoproteasome contains two outer rings composed of a-subunits and two internal 7-subunit containing rings each possessing 3 specific subunits responsible for proteasome catalytic activity. In the 20S immunoproteasome these subunits (b1, b2, b5) are replaced by three inducible subunits: PSMB9/LMP2, PSMB10/MECL1 and PSMB8/LMP7 (b1i, b2i, b5i). These stress-induced subunits allow for the production of MHC-1 associating peptides, which are displayed as antigens on the cell surface. These displayed peptides can then be recognized by immune surveillance CD8 T cells. The 20S immunoproteasome is recognized as a strong drug target for autoimmune disease and cancer. The 20S immunoproteasome is commonly associated with the 19S, PA28 a/b or the PA28g regulatory complexes.

Product Name PID Product description
20S Immunoproteasome (human) (untagged) SBB-PP0004 The 20S immunoproteasome is structurally similar to constitutive 26S proteasome. The 20S core of immunoproteasome contains two outer rings composed of α-subunits, and two internal 7-subunit containing rings each possessing 3 specific subunits responsible for proteasome catalytic activity. In immunoproteasomes these subunits (ß1, ß2, ß5) are replaced by three inducible subunits: PSMB9, PSMB10 and PSMB8, (ß1i, ß2i, ß5i). These stress-induced subunits allow for the production of MHC-1 associating peptides, which are displayed as antigens on the cell surface. These displayed peptides can then be recognized by immune surveillance CD8 T cells. 20S immunoproteasome is recognized as a strong drug target for autoimmune disease and cancer.
20S Proteasome (human) (untagged) SBB-PP0005 The ubiquitin-proteasome pathway is the major proteolytic system in eukaryotic cells, where it catalyzes the selective degradation of short-lived regulatory proteins or the rapid turnover of misfolded proteins. One of the most important proteases in this pathway is the 26S proteasome, an ATP-dependent proteolytic complex, which is formed by the association of the barrel-shaped 20S proteasome (700kDa) and two 19S (700kDa) regulatory complexes. The 20S catalytic core is composed of 4 rings of 28 non-identical subunits; 2 rings are composed of 7 α-subunits and 2 rings are composed of 7 β-subunits. The 20S catalytic core is able to degrade a variety of peptide substrates and poly-ubiquitinated proteins involved with apoptosis, DNA repair, endocytosis and cell cycle control.
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Ubiquitin (human) (rec.) (Rhodamine 110) Ubiquitin (human) (rec.) (Rhodamine 110) 50 µg Ref. SBB-PS0001-C050 Marque SOUTH BAY BIO 198.00 H.T.
 
ISG15 (human) (rec.) (Rhodamine 110) ISG15 (human) (rec.) (Rhodamine 110) 50 µg Ref. SBB-PS0002-C050 Marque SOUTH BAY BIO 302.00 H.T.
 
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NEDD8 (human) (rec.) (Rhodamine 110) NEDD8 (human) (rec.) (Rhodamine 110) 50 µg Ref. SBB-PS0003-C050 Marque SOUTH BAY BIO 302.00 H.T.
 
20S Immunoproteasome (human) (untagged) 20S Immunoproteasome (human) (untagged) 25 µg Ref. SBB-PP0004-C025 Marque SOUTH BAY BIO 222.00 H.T.
 
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20S Proteasome (human) (untagged) 20S Proteasome (human) (untagged) 50 µg Ref. SBB-PP0005-C050 Marque SOUTH BAY BIO 158.00 H.T.
 
Z-Leu-Leu-Glu-AMC [Z-LLE-AMC] Z-Leu-Leu-Glu-AMC [Z-LLE-AMC] 2 mg Ref. SBB-PS0006-M002 Marque SOUTH BAY BIO 124.00 H.T.
 
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Ac-Pro-Ala-Leu-AMC [Ac-PAL-AMC] Ac-Pro-Ala-Leu-AMC [Ac-PAL-AMC] 2 mg Ref. SBB-PS0007-M002 Marque SOUTH BAY BIO 144.00 H.T.
 
Ac-Trp-Leu-Ala-AMC [Ac-WLA-AMC] Ac-Trp-Leu-Ala-AMC [Ac-WLA-AMC] 2 mg Ref. SBB-PS0008-M002 Marque SOUTH BAY BIO 144.00 H.T.
 
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Ac-Ala-Asn-Trp-AMC [Ac-ANW-AMC] Ac-Ala-Asn-Trp-AMC [Ac-ANW-AMC] 2 mg Ref. SBB-PS0009-M002 Marque SOUTH BAY BIO 144.00 H.T.
 
Suc-Leu-Leu-Val-Tyr-AMC [Suc-LLVY-AMC] Suc-Leu-Leu-Val-Tyr-AMC [Suc-LLVY-AMC] 2 mg Ref. SBB-PS0010-M002 Marque SOUTH BAY BIO 63.00 H.T.
 
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p53 (polyubiquitinated) (human) (rec.) (His) p53 (polyubiquitinated) (human) (rec.) (His) 20 µg Ref. SBB-US0012-C020 Marque SOUTH BAY BIO 381.00 H.T.
 
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Ubiquitin (human) (rec.) (untagged) Ubiquitin (human) (rec.) (untagged) 10 mg Ref. SBB-UP0013-M010 Marque SOUTH BAY BIO 78.00 H.T.
 
Ubiquitin (human) (rec.) (Europium-Cryptate) Ubiquitin (human) (rec.) (Europium-Cryptate) 20 µg Ref. SBB-TR0014-C020 Marque SOUTH BAY BIO 653.00 H.T.
 
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Ubiquitin (human) (rec.) (Cy5) Ubiquitin (human) (rec.) (Cy5) 50 µg Ref. SBB-TR0015-C050 Marque SOUTH BAY BIO 183.00 H.T.
 
Ubiquitin (human) (rec.) (6-FAM) Ubiquitin (human) (rec.) (6-FAM) 50 µg Ref. SBB-TR0016-C050 Marque SOUTH BAY BIO 183.00 H.T.
 
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UBE2D3 [UbcH5c ] (human) (rec.) (untagged) UBE2D3 [UbcH5c ] (human) (rec.) (untagged) 100 µg Ref. SBB-CE0019-C100 Marque SOUTH BAY BIO 236.00 H.T.
 
UBE2L3 [UbcH7] (human) (rec.) (untagged) UBE2L3 [UbcH7] (human) (rec.) (untagged) 100 µg Ref. SBB-CE0020-C100 Marque SOUTH BAY BIO 236.00 H.T.
 
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UBE2D1 [UbcH5a] (human) (rec.) (untagged) UBE2D1 [UbcH5a] (human) (rec.) (untagged) 100 µg Ref. SBB-CE0021-C100 Marque SOUTH BAY BIO 236.00 H.T.
 
UBE2K [UbcH1, E2-25K] (human) (rec.) (His) UBE2K [UbcH1, E2-25K] (human) (rec.) (His) 100 µg Ref. SBB-CE0022-C100 Marque SOUTH BAY BIO 236.00 H.T.
 
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UcH-L3 (human) (rec.) (untagged) UcH-L3 (human) (rec.) (untagged) 50 µg Ref. SBB-DE0023-C050 Marque SOUTH BAY BIO 147.00 H.T.
 
PLpro (SARS Coronavirus) (rec.) (His) PLpro (SARS Coronavirus) (rec.) (His) 50 µg Ref. SBB-DE0024-C050 Marque SOUTH BAY BIO 197.00 H.T.
 
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NEDP1 [SENP8] (human) (rec.) (His) NEDP1 [SENP8] (human) (rec.) (His) 50 µg Ref. SBB-DE0025-C050 Marque SOUTH BAY BIO 147.00 H.T.
 
SENP1 (catalytic domain) (human) (rec.) (His) SENP1 (catalytic domain) (human) (rec.) (His) 50 µg Ref. SBB-DE0026-C050 Marque SOUTH BAY BIO 147.00 H.T.
 
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UBE2D2 [UbcH5b] (human) (rec.) (untagged) UBE2D2 [UbcH5b] (human) (rec.) (untagged) 100 µg Ref. SBB-CE0027-C100 Marque SOUTH BAY BIO 204.00 H.T.
 
SUMO1 (human) (rec.) (Rhodamine 110) SUMO1 (human) (rec.) (Rhodamine 110) 50 µg Ref. SBB-PS0028-C050 Marque SOUTH BAY BIO 302.00 H.T.
 
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SUMO2 (human) (rec.) (Rhodamine 110) SUMO2 (human) (rec.) (Rhodamine 110) 50 µg Ref. SBB-PS0029-C050 Marque SOUTH BAY BIO 302.00 H.T.
 
Ubiquitin aldehyde (human) (rec.) Ubiquitin aldehyde (human) (rec.) 50 µg Ref. SBB-PS0031-C050 Marque SOUTH BAY BIO 108.00 H.T.
 
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Ubiquitin vinyl sulfone (human) (rec.) Ubiquitin vinyl sulfone (human) (rec.) 50 µg Ref. SBB-PS0032-C050 Marque SOUTH BAY BIO 193.00 H.T.
 
Ubiquitin vinyl methyl ester (human) (rec.) Ubiquitin vinyl methyl ester (human) (rec.) 50 µg Ref. SBB-PS0033-C050 Marque SOUTH BAY BIO 223.00 H.T.
 
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Ubiquitin-propargylamide (human) (rec.) Ubiquitin-propargylamide (human) (rec.) 50 µg Ref. SBB-PS0034-C050 Marque SOUTH BAY BIO 178.00 H.T.
 
MDM2 E3 Ligase TR-FRET Kit MDM2 E3 Ligase TR-FRET Kit 1 Kit Ref. SBB-KF0030-KI01 Marque SOUTH BAY BIO 678.00 H.T.
 
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ITCH E3 Ligase TR-FRET Kit ITCH E3 Ligase TR-FRET Kit 1 Kit Ref. SBB-KF0035-KI01 Marque SOUTH BAY BIO 678.00 H.T.
 
Parkin E3 Ligase TR-FRET Kit Parkin E3 Ligase TR-FRET Kit 1 Kit Ref. SBB-KF0036-KI01 Marque SOUTH BAY BIO 678.00 H.T.
 
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TRF-Ubiquitin Mix (100X) (2000 reactions) TRF-Ubiquitin Mix (100X) (2000 reactions) 1 Vial Ref. SBB-TR0051-2K-1 Marque SOUTH BAY BIO 564.00 H.T.
 
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20S Immunoproteasome Assay Kit 20S Immunoproteasome Assay Kit 1 Kit Ref. SBB-KP0037-KI01 Marque SOUTH BAY BIO 495.00 H.T.
 
20S Constitutive Proteasome Assay Kit 20S Constitutive Proteasome Assay Kit 1 Kit Ref. SBB-KP0038-KI01 Marque SOUTH BAY BIO 389.00 H.T.
 
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Nedd4 E3 Ligase TR-FRET Kit Nedd4 E3 Ligase TR-FRET Kit 1 Kit Ref. SBB-KF0056-KI01 Marque SOUTH BAY BIO 678.00 H.T.
 
XIAP E3 Ligase TR-FRET Kit XIAP E3 Ligase TR-FRET Kit 1 Kit Ref. SBB-KF0057-KI01 Marque SOUTH BAY BIO 678.00 H.T.
 
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