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Summer 2009

FEATURED CONTENT:ubiquitin chains• sumo chains • affinity matrices (uim,sim)

www.bostonbiochem.com

UBIQUITIN CHAINS
The type and number of poly-ubiquitin chains that are conjugatedto a target is highly regulated to generate distinct signals that affectdifferent physiological processes. This versatility arises fromthe fact that not only can targets be mono-ubiquitinated or
poly-ubiquitinated, but also that different types of poly-ubiquitin chainsare formed. Proteins tagged with ubiquitin are most often destined fordegradation by the proteasome. However, mono-ubiquitination andpoly-ubiquitination also has non-proteasomal regulatory functions liketargeting proteins to nucleus, cytoskeleton and endocytic machinery, ormodulating enzymatic activity and protein-protein interactions.
Multi-ubiquitin chains are built by formation of an isopeptide bond
between Gly76 of one ubiquitin to the ε-NH2 group of one of theseven potential lysines (K6, K11, K27, K29, K33, K48 or K63) of thepreceding ubiquitin. Specific E2 enzymes and E2/E3 combinations result in the formation of linkage-specific ubiquitin chains.
Residue K48 is a major site of chain initiation and K48-linkages are highly abundant, being the predominant signal for proteins
destined for degradation by the proteasome. The other principle and relatively abundant poly-ubiquitin chain has K63-linkages.
K63-linkages do not seem to play a role in protein turnover and have been implicated in receptor endocytosis and sorting,translation, DNA damage repair, the stress response and signaling.

Figure 1. K48-linked ubiquitin chain mixtures (Ub1-7, UC-240), with monoUb removed (Ub2-7, UC-230), and with both mono- and di-Ub removed (Ub3-7,UC-220).

Figure 2. Deconjugation of K63-linked ubiquitin chains by Isopeptidase
T to generate mono-ubiquitin. Chain species (4μg) were incubated with enzyme for 30 minutes at 37٥C in 50 mM Hepes pH 8.0, 100 mM NaCl, 5mM
DTT. (Ub2,UC-300; Ub4,UC-310; Ub3,UC-315; Ub5, UC-316)

Ubno Ub

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UBIQUITIN CHAINS• Linkages

K48 Di-Ub Chains

Lysine 48 (K48), Lysine 63 (K63) or linear fusion• Lengths

From mono Ub to high molecular weight chains• Bulk Quantities• Custom Ub Chains

Using affinity tagged, labeled and/or mutant Ub toform chains with different linkages and lengths

K48-Linked Chains:
Di-Ub WT Chains (Ub2)

Catalog No.

Size

UC-200100 μg

Tetra-Ub WT Chains (Ub4)UC-21025 μg

Tetra-Ub WT Chains(Ub-K63-Ub-K48-Ub-K63-Ub)

UCM-21025 ug

Tri-Ub WT Chains (UB3)

UC-215100 µgPenta-Ub WT Chains (Ub5)UC-21625 μgPoly-Ub WT Chains (Ub3-7)

UC-220100 μgPoly-Ub WT Chains (Ub2-7)

UC-230100 μgHis6-Poly-Ub WT Chains (Ub2-7)

UCH-230

100 µgPoly-Ub WT Chains (Ub1-7)

UC-240100 μg

K63 Di-Ub Chains

66 kDa55 kDa66 k
55 k

36 kDa31 kDa36 k
31 k

21 kDa21 k

14 kDa

di Ub

6 kDa

mono Ub

K63-Linked Chains:

Catalog No.

50 μg

Tetra-Ub WT Chains (Ub4)UC-31025 μg

Tetra-Ub WT Chains(Ub-K48-Ub-K63-Ub-K48-Ub)

UCM-31025 ug

Tri-Ub WT Chains (UB3)

UC-315100 μgPenta-Ub WT Chains (Ub5)UC-31625 μg

Hexa-Ub WT Chains (Ub6)UC-31725 μg

UC-31825 μgPoly-Ub WT Chains (Ub3-7)

UC-320100 μgPoly-Ub WT Chains (Ub2-7)

36 kDa31 kDaPoly-UbWT Chains (Ub )UC-330100 μgHis6-Poly-Ub WT Chains (Ub2-7)

UCH-330

100 µg

UC-340100 μgOther Linkages:

Catalog No.

Size

UC-700100 μg

21 k

di Ub

14 k

1-7

Linear(Ub2)
21Di-UbiquitinkDa

14 kDa21 kDa

6 kDa

14 kDa

di Ub

6 kDa

mono Ub

Figure 3. Hydrolysis of K63-linked Ub chains by DUBs. DUBs wereincubated with 4μg di-ubiquitin linked through K63 (UC-300).

Mixed Tetra: Ub-K63-Ub-K48-UbK63

Size

Di-Ub WT ChainsMixed(Ub2) Tetra: Ub-K48-Ub-K63-UbK48
UC-30055 kDa36 kDa31 kDa6k

Figure 4. Hydrolysis of K48-linked Ub chains by DUBs. DUBs wereincubated with 4μg di-ubiquitin linked through K48 (UC-200).

66 kDaOcta-UbWT Chains (Ub8)

66 kDa55 kDa14 kmono Ub

Linkage specific poly-Ubiquitin chains are used to investigate mechanisms of binding and recognition by E1 activating or E2 conjugating enzymes, deubiquitinating enzymes, E3 ligases or other proteins that contain ubiquitininteracting motifs (UIMs). Chain products are formed
with wild-type ubiquitin and linkage-specific conjugating
enzymes.

For additional information visit www.bostonbiochem.com or call 617-241-7072

66 k
55 k

36 k
31 k

6k

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SUMO CHAINS
Similar to ubiquitin, the small ubiquitin-related modifier (SUMO) canform polymeric chains on many of its targets, and the poly-SUMO signal
is responsible for a number of specific biological functions. Similar toubiquitination, SUMOylation is a reversible process performed by SUMO-specific proteases (SENPs) which function in both the maturation of
SUMO precursor proteins, the disassembly of SUMO chains, and in theremoval of SUMO from modified substrates.
We offer a range of polymeric SUMO chains to investigate the substratespecificity of SENP enzymes. These chains can also be used to investigate the mechanism of binding and recognition by SUMO specific E1or E2 enzymes, E3 ligases or other proteins that contain SUMO binding
domains.

• SUMO-2 and SUMO-3 Chains• Bulk Quantities• Custom Chain Synthesis

SUMO Chains

Catalog No.

Size

di-SUMO-2 WT Chains

ULC-200

50 µg

Poly-SUMO-2 WT Chains (2-8)

ULC-21025 µg

Poly-SUMO-2 WT Chains (3-8)

ULC-22025 µg

di-SUMO-3 WT Chains

ULC-300

50 µg

Poly-SUMO-3 WT Chains (2-8)

ULC-31025 µg

Poly-SUMO-3 WT Chains (3-8)

ULC-320

25 µg

Figure 1. In vitro mediated SUMO-2/3 chain formation is abrogated by the mutationof the conserved lysine 11 to arginine. SUMO chains were synthesized in the presence of
SUMO E1 (E-315) and UbcH9 (E2-645) enzymes. Reaction products were visualized by
16% SDS-PAGE.

SUMO Mutants

Catalog No.

Size

SUMO-1 K7R

ULM-710250 µg

SUMO-1 K16R

ULM-712250 µg

SUMO-1 K17R

ULM-714250 µgFigure 2.
In vitro
mediated SUMO-2/3 chain formation is abrogated
SUMO-1
K7R,
K16R
ULM-716by the mu250 µg
tation of the conserved lysine 11 to arginine (SUMO-2 K11R, ULM-752; SUMO-3SUMO-1
K7R,
K17R
ULM-718
K11R, ULM-762). SUMO chains were synthesized in the presence of SUMO250
E1 µg(E-311) and UbcH9 enzymes (E2-645). Reaction products were visualized by 16%

SUMO-1 K16R, K17R

ULM-720250 µg

SUMO-1 K7R, K16R, K17R

ULM-722250 µg

SUMO-2 K11R

ULM-752

250 µg

SUMO-3 K11R

ULM-762

250 µgFigure 2. Deconjugation of SUMO-2 and SUMO-3 chains by SUMO-specificisopepidases SENP1 and SENP2. Reactions contained 2μg of SUMO chains(Di-SUMO-2, ULC-200; SUMO-22-8, ULC-210; SUMO-23-8, ULC-220; Di-SUMO-3,ULC-300; SUMO-32-8, ULC 310; SUMO-33-8, ULC-320) in 50mM Hepes, 50mM
NaCl, 5mM DTT and were incubated in the presence of 500nM SENP1 (S1)(E-700) orSENP (S2)(E-710) for 60 minutes at 37٥C.

Figure 3. SUMOylation motif (ΨKxE) is underlined. The precursor forms for each SUMO contains the residues beyond the conserved
C-terminal GG. Conserved lysines that play a critical role in SUMO polymerization are indicated as (K). SUMO-1 contains a total of 11lysine residues, with some (K) not found in SUMO-2 and SUMO-3, which both have a total of 7 lysines. Primary sequence alignment ofSUMO-1, SUMO-2 and SUMO-3 proteins. Residues in blue are identical, to the consensus

For additional information visit www.bostonbiochem.com or call 617-241-7072

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AFFINITY MATRICES (UIM, SIM)
Affinity matrices are resins with covalently attached peptides or proteins and are excellent tools for the enrichment, isolation and identification of specific interacting proteins. Boston Biochem offers a variety of matrices which can specifically identify ubiquitin and SUMO
chains, and also ubiquitinated or SUMOylated protein substrates.
Our reagents have the highest protein:agarose coupling ratios available to maximize binding for low abundance or low affinity proteinsof interest.
SIM Agarose with SUMO Chains
SUMO22-7 SUMO32-7
Input

Bound BoundSIM Agarose with Ub Chains
K48-linked1-7

K63-linked1-7

Input Bound

Input Bound

S6S5
S4
S3

S2

Ub 7
Ub 6
Ub 5

Ub 7
Ub 6
Ub 5
Ub 4

Ub 4

Ub 3

Ub 3

Ub 2

Ub 2

Ub 1

Ub 1

Ataxin UIM-agarose

AM-1150.25 mlAtaxin-3 has deubiquitinating activity in vitro. The full-length protein contains a catalytic N-terminal Josephin domain, three ubiquitin interacting motifs (UIMs), anda variable C-terminus with a polyglutamine stretch. Ataxin3 functions as a mixed
linkage, chain editing enzyme with preferential cleavage of K63 linkages in mixed
chains. Ataxin3 also binds both K48-linked and K63-linked poly-Ub chains via its
UIM domains. This affinity resin can be used for the enrichment, isolation and identification of K48-linked or K63-linked poly-Ub chains or ubiquitinated substrates that
contain these linkages.

Rap80 UIM-agarose

AM-1200.25 ml
Rap80 (Receptor Associated Protein 80) interacts with BRCA1, a Ub E3-ligase whichfunctions in conjugation with the BARD1 deubiquitinating enzyme. BRCA1 is recruited to DNA damage sites by poly-ubiquitin chains through Rap80 which contains 2
tandem ubiquitin-interacting motifs (UIMs). The UIM domains of Rap80 have beenshown to have preferential binding to K-6 and K63-linked Ub chains. Rap80 hasa low affinity for mono-, di- and tri-Ub but binds efficiently to tetra (or greater) Ubchains. This affinity resin can be used for the enrichment, isolation and identification of ubiquitinated proteins that are non-K48-linked.

SUMO-interacting Motif (SIM) Peptide Agarose

Figure 1. SUMO Interacting Motif (SIM) Agarose binds specifically to SUMOchains. A mixture of either Ub (20μg) (K48 Ub1-7, UC-240; K63 Ub1-7, UC-340) orSUMO(10μg) chains was incubated with 15μl of SIM agarose (AM-200) for 1.5 hours
at room temperature. The resin was washed 3 times for 1 minute each with 1ml of washbuffer. The resin was resuspended in 30μl SDS-PAGE sample buffer and heated for 2
minutes at 37°C, and 10μl of supernatant was loaded on a 16% SDS-PAGE gel.

AM-2000.5 mlThree different amino acid consensus motifs have been identified as mediating
SUMO binding and/or interaction. These SUMO-interacting motifs (SIMs) all contain a hydrophobic core sequence with a stretch of acidic amino acids either at theN- or C-terminus. Studies indicate that the SIM hydrophobic region is essential for
mediating binding to the α-helix and β2-strand surfaces on SUMO proteins. The negatively charged residues surrounding the hydrophobic core can influence binding
affinities and can dictate binding preferences for the various SUMO isoforms. Thisaffinity resin is derived from a PIAS sequence, and can be used for the enrichment,
isolation and identification of SUMOylated proteins.

S5a Agarose
AM-1000.5 ml
S5a (Rpn10) is a component of the regulatory complex (19S) of the 26S proteasome. It binds to and recognizes poly-ubiquitinated proteins and functions as areceptor for proteins destined for proteolytic degradation. The protein recognizes
Ub chains and conjugates via two Ub-interacting motif (UIM) domains. S5a has
a preference for longer Ub polymers and has a low affinity for mono-, di- and triUb. This affinity resin can be used for the enrichment, isolation and identificationof ubiquitinated proteins, 26S substrates or proteins that contain Ub-like domains.

S5a UIM Peptide Agarose AM-110

0.5 ml

S5a (Rpn10) is a component of the regulatory complex (19S) of the 26S proteasome.
It binds to and recognizes poly-ubiquitinated proteins and functions as a receptor forproteins destined for proteolytic degradation. The protein recognizes Ub chains andconjugates via two Ub-interacting motif (UIM) domains located at residues 211-230(I) and 282-301 (II). Although both UIMs bind to poly-ubiquitin in vitro, UIM II has
a 10-fold higher affinity for ubiquitin than UIM I. This affinity resin can be used forthe enrichment, isolation and identification of ubiquitinated proteins, proteins that
contain ubiquitin-like domains and/or 26S substrates.

Figure 2. Preferential binding of K63- and K48-linked Ub chains to Rap80 UIM andAtaxin UIM agarose, respectively. A mixture of K63- or K48-linked Ub chains (10μg)
was incubated with 25μl Rap80 (AM-120) or Ataxin UIM (AM-115) agarose in a 100μl
volume for 1 hour at room temperature. The resin was washed 5 times for 1 minute eachwith 1ml buffer. The resin was resuspended in 20μl SDS-PAGE sample buffer, 12μl of thesupernatant and 2μg of the initial Ub chain input was separated on a reduced 16% SDSPAGE gel.

Boston Biochem, Inc.

840 Memorial DriveCambridge, MA 02139
T 617-241-7072
F 617-492-3565
E customerservice@bostonbiochem.com

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