System IMPACT_CN.pdf

Tools for

Protein Research

GENESEARCH

the leader in enzyme technology

Jan. 2005

Contents

Peptide-Carrier ™ Kit

◆ Peptide-Carrier Kit.................1

◆ Protein Expression Kits...........2

◆ Proteomics ..............................3

◆ Protein Markers ......................3

◆ Magnetic Matrices ..................4

◆ Other Protein Tools ............... 4

The Peptide-Carrier Kit allows peptides of interest

to be detected on peptide arrays (Figure 1) or

Western blots (Figure 2) through ligation to a

carrier protein. The resulting peptide-carrier

ligation product is compatible with a variety of

applications including Western blotting, peptide

arrays and kinase assays (Figure 3). Simply mixing

a peptide (N-terminal cysteine required) with the

supplied Carrier Protein 39 (CP39, ~39 kDa)

results in a peptide-carrier ligation product via

intein-mediated protein ligation (IPL).

Since most antigenic peptides are routinely synthe-

sized with a cysteine, a single peptide containing

an N-terminal cysteine can now be used to gener-

ate antibodies as well as a control for Westerm

blots. The antigenic peptide may contain modified

residues, such as a phosphorylated tyrosine. The

ligated product has one antigenic peptide per

CP39 resulting in a single, sharp band on Western

blots (Figure 2).

This kit also makes it possible to produce peptide

substrates for enhanced sensitivity in array analysis

(Figure 1). In addition, peptide substrates ligated to

Carrier Proteins 39 or 27 can be used for

enzymatic assays and subsequent Western blot

analysis (Figure 3).

kDa

1 2 3 4

kDa

1 2 3 4

Genesearch is the exclusive Australian

agent for:

New England Biolabs – enzymes &

reagents for molecular biology

Cell Signaling Technology – activation state

specific antibodies

Gene Codes – DNA sequence assembly &

analysis software (Sequencher)

hrs:

C .25 1 16

4 4 4 4

°C:

4 4 4 4

Figure 2: Analysis of peptide antigen PB1

ligated to Carrier Protein CP39.

A) Western blot analysis of antiserum raised

against PB1. PB1 was synthesized with a

N-terminal cysteine (CTRSRHSSYPNEY-

EEDEEMEEEL; MW 2862 Da) and ligated to

Carrier Protein 39 (Western) (NEB #E6602).

The samples were electrophoresed on a

12% SDS polyacrylamide gel and

transferred to nitrocellulose. Anti-PB1

Peptide Antibody (NEB #E6608) was used

as the primary antibody. Lane 1 is a control

(Western).

B) Analysis of peptide ligation by Coomas-

sie blue stained SDS-PAGE. The ligation

samples were electrophoresed on a 12%

SDS polyacrylamide gel and examined by

Coomassie blue staining. Lane 1 is a

control (C) which contains only CP39.

HA tag P1 CAGAG A P Y D V P D Y A

HA tag P2 CAGAG Y A Y D V P D Y A

HA tag P3 CAGAG Y P A D V P D Y A

HA tag P4 CAGAG Y P Y A V P D Y A

HA tag P5 CAGAG Y P Y D A P D Y A

HA tag P6 CAGAG Y P Y D V A D Y A

HA tag P7 CAGAG Y P Y D V P A Y A

HA tag P8 CAGAG Y P Y D V P D A A

HA tag P9 CAGAG Y P Y D V P D Y A

P1 P2 P3 P4 P5 P6 P7 P8 P9

Peptide

(nmol)

Applications:

* Generation of positive controls for Western blot

analysis

* Production of substrates for enhanced sensitiv-

ity in peptide arrays or dot blot assays

* Production of substrates for protein modifica-

tion

* Ligation of a peptide containing natural or

unnatural amino acids

The Kit Includes:

* Carrier Protein 39 (Western): 50 reactions

* Control Peptide, PB1: 50 µl

* Carrier Protein (CP) Reaction Buffer: 0.5 ml

* SDS Sample Buffer: 0.5 ml

* Detailed Instruction Manual

#E6600S 50 reactions $344

3.33

1.11

0.37

0.12

3.33

1.11

0.37

CP39-HA

kDa

CP39-peptide

+ Abl Kinase

0.12

Figure 1: Alanine scan of the hemagglutinin

(HA) epitope.

A) HA peptide library was synthesized with a N-

terminal cysteine. P9 contains the wild type

sequence corresponding to residues Tyr 98 to Ala 106

of the hemagglutinin (HA) protein. Each of the

other 8 peptides (P1-P8) carries a single substitu-

tion with an alanine residue.

B) Each peptide was ligated to Carrier Protein 39

(Array) (NEB #E6603) overnight at 4°C. The ligated

products (rows 1–4) were arrayed on a 0.45 µm

nitrocellulose membrane along with the unligated

peptides (rows 5-8). The blot was reacted with an

anti-HA monoclonal antibody.

The data indicates that the residues mutated in P1,

P2, P4 and P5 are essential for antibody recogni-

tion and the unligated peptide (rows 5–8) did not

produce a detectable signal.

1 2 3 4 5 6

Figure 3: Kinase Assay.

A peptide containing a candidate

phosphorylation site was synthesized with

an additional N-terminal cysteine (CGS-

NEAIYAAPFAKKK; 7) and ligated to CP39.

The ligated product was phosphorylated

with Abl Protein Tyrosine Kinase (NEB

#P6050) and then subjected to Western

blot analysis using anti-phospho-tyrosine

antibody (Cell Signaling Technology). A

positive signal was detected only in lanes 3

and 4 where the ligated product was

treated with Abl Kinase. Lane 1: CP39.

Lane 2: CP39 + Abl Kinase. Lane 3: CP39-

peptide + Abl Kinase (50 units). Lane 4:

CP39-peptide + Abl Kinase (100 units).

Lane 5: CP39-peptide. Lane 6: Abl Kinase.

Advantages

* Easy to use – just mix the components

* Optimised for Western blots & arrays

* Quick – generate substrate within an

hour

* Deﬁned position with 1:1 stoichiometry

* Non-radioactive

* Inexpensive

* Versatile – one peptide for multiple

applications; easily incorporate modi-

ﬁed (P-, Ac-, Me-) residues

* You are in control of target information

and production of array

Introductory Offer!

References

Xu et al. (2004) Biotechniques 36:976-981

Sun et al. (2004) Biotechniques 37:430-443

20% off list price until 31 March 2005

= Less than $6 per reaction

One per lab; Conditions apply

Genesearch Pty Ltd Tel: 1800 074 278 Fax: 07 5594 0562 Email: info@genesearch.com.au Web: www.genesearch.com.au

Protein Expression & Purification

Protein Fusion Systems

IMPACT ™ Systems

IMPACT ™ (Intein Mediated Purification with an Affinity Chitin-binding Tag)

utilizes the inducible self-cleavage activity of engineered protein splicing elements

(termed inteins) to purify recombinant proteins by a single affinity column. Unlike

other protein fusion systems, you can separate a recombinant protein from the

affinity tag without the use of a protease.

The IMPACT-CN Protein Fusion and Purification System contains 4

expression vectors (pTYB vectors) which allow fusion of a bifunctional tag, consist-

ing of the intein and the chitin binding domain, to either the C-terminus or N-

terminus of the target protein. In the presence of thiols such as DTT, ß-

mercaptoethanol or cysteine, the intein undergoes specific self-cleavage which

releases the target protein from the chitin-bound intein tag.

Advantages:

* Flexibility - allows fusion to either the C-terminus or N-terminus of the target

protein

* Yields proteins with native sequence

* Release of fusion partner without the use of proteases

* One-step affinity purification-no additional steps to remove affinity tag

* T7 Promoter for higher levels of expression

* Tight transcriptional control

* The ability to label the C-terminus of the target protein

#E6900S 1 kit $826

The IMPACT ™ -TWIN System uses mini-inteins for protein purification &

manipulation. This unique system can produce proteins with an N-terminal cysteine

and/or C-terminal thioester for use in protein labeling, ligation & cyclisation.

#E6950S

C-terminal Fusion

N-terminal Fusion

N-terminal C-terminal

Target Gene

T7 Promoter

MCS Intein Tag

Intein Tag

MCS

T7 Promoter

Target

Protein

Intein

Tag

Intein

Tag

Target

Protein

Load &

Wash

Chitin

Inducible Cleavage

+DTT at 4 ° C

Elute

Elute &

Dialyze

Target

Protein

Target

Protein

Schematic illustration of the IMPACT -CN System.

1 kit $826

pMAL ™ System

In the pMAL ™ Protein Fusion and Purification System, the cloned gene is inserted

into a pMAL vector down-stream from the malE gene, which encodes maltose-

binding protein (MBP), resulting in the expression of an MBP-fusion protein. The

technique uses the strong Ptac promoter and the translation initiation signals of

MBP to express large amounts of the fusion protein. The fusion protein is then

purified by a one-step affinity purification specific for MBP.

The system uses the pMAL vectors which are designed so that insertion interrupts a

lacZ α gene allowing a blue-to-white screen for inserts on X-gal. The vectors include

a sequence coding for the recognition site of a specific protease. This allows the

protein of interest to be cleaved from MBP after purification, without adding any

vector-derived residues to the protein. For this purpose, the polylinker includes a

restriction site superimposed on the sequence coding for the site of the specific

protease. This is where the gene of interest is inserted.

Expression from the pMAL vectors yields up to 100 mg fusion protein from a liter of

culture. In most cases, the expressed protein is soluble, as fusion to MBP has been

proven to enhance the solubility of proteins expressed in E. coli . While no expression

system works with every cloned gene, the pMAL ™ Protein Fusion and Purification

System gives substantial yields of protein in more than 75% of the cases tested.

Advantages:

* Reliable expression: substantial yields (up to 100 mg/L) in more than 75% of the

cases tested.

* Expression in either the cytoplasm or periplasm: periplasmic expression enhances

folding of proteins with disulfide bonds.

* Fusion to MBP has been shown to enhance the solubility of proteins expressed in

E. coli .

* Gentle elution with maltose: no detergents or harsh denaturants.

#E8000S

Target Gene

mal E

MCS

lacZ α

Cloning &

Expression

Protease

Cleavage

Site

MBP

Target

Protein

Load & Wash

Amylose

MBP

Target

Protein

Elute With Maltose

MBP

Target

Protein

Specific Protease

MBP

Target

Protein

1 kit $826

Schematic illustration of the pMAL System.

Cleave With

For PROTEOMICS

Proteases

NEB’s proteases for proteomics are free of glycerol

and detergents which may interfere with Matrix-

assisted laser desorption/ionization time-of-flight

(MALDI-TOF) Mass Spectrometry (MS) or liquid

chromatography (LC) methods.

Applications:

* Digestion of proteins for proteomic analysis by

Mass Spectroscopy

* Protein & peptide identification

MALDI-TOF analysis of myoglobin in

solution digest with GluC

Peptide Standards

A set of five peptides which can be used to

calibrate a Matrix-assisted laser desorption/

ionization time-of-flight (MALDI-TOF) or

Electrospray Ionization (ESI) mass spectrom-

eter. Free of salts, glycerol and detergents.

#P7720S

Myoglobin (whale) was digested with a 1:10 ratio of recombinant GluC to

protein in 10 mM Tris-HCl pH 8

100

2857.02

1449.43

1482.42

2317.02

10 µg each $114

ACTH (18-39)

2204.43

100

1650.49 2547.79

Angiotensin

Endoproteinase GluC

Endoproteinase GluC ( Staphylococcus aureus Protease

V8) is a serine proteinase which selectively cleaves

peptide bonds C-terminal to glutamic acid residues.

Endoproteinase GluC also cleaves at aspartic acid

residues at a rate 100-300 times slower than at

glutamic acid residues.

Recombinant: higher purity at lower cost!

#P8100S

ACTH (1-17)

1694.58

3340.65

1004.0 1532.8 2061.6 2590.4 3119.2 3648.0

Neurotensin

Mass (m/z)

ACTH (7-38)

Peaks from myoglobin: Peaks from GluC:

M/z theoretical residues M/z theoretical residues

868.86 869.9 *

19-27 1650.49 1649.89

42-54

1449.43 1449.7 42-52

1482.42 1482.76 137-148

1694.58 1694.58 5-18/ 7-20

2204.43 2204.49 19-38

2317.02 2316.79 86-105

2547.79 2547.87 19-41

2857.02 2857.21 110-136

3340.65 3341.94 55-85/ 53-83

* Present in original but not shown

in above chart

750.0

1485.8

2221.6

2957.4

3693.2

4429.0

Mass (m/z)

MS Protein Standards

A set of five proteins which can be used to

calibrate a Matrix-assisted laser desorption/

ionization time-of-flight (MALDI-TOF) or

Electrospray Ionization (ESI) mass spectrom-

eter. Free of salts, glycerol and detergents.

#P7730S 0.5 nmol each $114

50 µg $114

The reaction was incubated overnight at room temperature and spotted directly on a MALDI

target. The matrix used here was α-cyano-4-hydroxycinnamic acid. The theoretical digests

were generated using the Peptide Mass software from ExPASy.

Modified Trypsin (TPCK-treated)

Modified Trypsin (TPCK-treated) is a serine

endopeptidase. It selectively cleaves peptide bonds

C-terminal to lysine and arginine residues.

Modified Trypsin is treated with L-(tosylamido-2-

phenyl) ethyl chloromethyl ketone (TPCK) to

inactivate any remaining chymotryptic activity, and

modified by acetylation of the ε-amino groups of

lysine residues to prevent autolysis. Modified

Trypsin cleaves at Lys-Pro and Arg-Pro bonds at a

much slower rate than other amino acid residues.

#P8101S

MALDI-MS analysis of cytochrome c digest

with Modified Trypsin

Yeast Cytochrome c was digested with a 1:20 ratio of Modified-trypsin to

protein in 50 mM Tris-HCl pH 8, 20 mM CaCl 2

100

2204.13

1782.73

1197.17

2065.44

Protein Monoisotopic Average MW

MW (daltons) (daltons)

RNAse A (Bovine) 13,681.32 13,690.29

Triose Phosphate Isomerase ( E.coli ) 26,954.82 26,971.81

Bovine Serum Albumin (BSA) 66,419.87 66,462.98

Phosphorylase b (rabbit) 97,096.82 97,158.22

MBP-ß-galactosidase fusion ( E.coli ) 158,095.29 158,194.87

2323.40

1654.63

1031.91

1383.85

100 µg $114

694.0 1107.2 1520.4 1933.6 2346.8 2760.0

Mass (m/z)

Protein Labeling

These synthetic peptides containing an N-terminal

cysteine and a biotinylated or fluorescein-labeled

lysine can be used in conjunction with the

IMPACT-TWIN (NEB #E6950S) or IMPACT-CN

(NEB #E6900S) Kits to site-specifically incorporate

a biotin or fluorescein moiety into a bacterially-

expressed protein. The peptide ligates to the C-

terminus of proteins containing a C-terminal

thioester via “intein-mediated protein ligation”

(IPL). The thioester-tagged proteins are purified

using either the IMPACT-TWIN or IMPACT-CN

Systems in which, preferably, 2-mercaptoethane-

sulfonic acid (MESNA) induces intein cleavage.

The peptide sequence is NH 2 -CDEPEK(*)DS-

COOH where K(*) represents a lysine with biotin or

fluorescein covalently attached to its ε-amino group.

Applications:

* Site-specifically labeling the C-terminus of a

protein with biotin or fluorescein

Peaks from cytochrome c: Peaks from Trypsin:

M/z theoretical residues M/z theoretical residues

1031.91 1031.51 1-10 2204.13 2197.06 50-69

1163.10 1159.61 1-11

1197.17 1196.65 34-44

1654.63 1654.81 96-109

1782.73 1783.80 45-60

2323.40 2321.12 60-79

2323.40 2326.15 12-33

Protein Markers

Protein Marker, Broad Range is a

mixture of purified proteins with

known amino acid sequences. They are

resolved to 13 sharp bands (2-212 kDa)

when analyzed by SDS-PAGE (Tris-

Glycine) and stained with Coomassie

Blue R-250. Two bands (BSA, MW

66.4 kDa and Triosephosphate isomer-

ase, MW 26.6 kDa) are at double

intensity to serve as reference points.

#P7702S 150 mini-gel lanes $124

#P7702L 750 mini-gel lanes $496

– 212

– 158

– 116

– 97.2

– 66.4

– 55.6

– 42.7

– 34.6

kDa

The reaction was incubated overnight at room temperature and spotted directly on a MALDI

target. The matrix used here was α-cyano-4-hydroxycinnamic acid. The theoretical digests

were generated using the Peptide Mass software from ExPASy.

Chitin

Step 1: N-S Shift

– 27.0

Target Protein

Intein-tag

Cys

– 20.0

Step 2: Thiol Mediated

Cleavage

3 2 2

SO -CH -CH -SH

H 2

– 14.3

– 6.5

– 2.4

Target Protein

Intein-tag

Cys

10–20%

SDS-PAGE

Step 3: Peptide Attack

Prestained Protein Marker,

Broad Range is a mixture of purified

proteins covalently coupled to a blue

dye that resolves to 8 bands of 6-175

kDa when electrophoresed. The

protein concentrations are carefully

balanced for even intensity. The

covalently coupled dye affects the

proteins’ electrophoretic behavior in

SDS-PAGE gels relative to unstained

proteins. For precise molecular weight

determinations, use the unstained

Protein Marker, Broad Range in

addition to the prestained marker.

#P7708S 175 mini-gel lanes $177

#P7708L 875 mini-gel lanes $708

– 175

kDa

NH 2

Target Protein S-CH -CH -SO2 -

Bio-P1

– 83

– 62

Cys

Step 4: S-N Shift

NH 2

– 47.5

Target Protein

Bio-P1

Cys

Bio-P1 (Lyophilized)

For labeling proteins with biotin.

#P6607S

– 32.5

– 25

Target Protein

Bio-P1

0.29 mg $197

Cys

Flu-P1 (Lyophilized)

For labeling proteins with fluorescein.

#P6606S

Figure: Intein-mediated Protein Ligation

– 16.5

– 6.5

0.29 mg $197

10–20%

SDS-PAGE

Prices shown throughout this brochure do not include GST. Prices are correct at time of publication but subject to change:

See www.genesearch.com.au/prices for current prices.

Magnetic Affinity Matrices

Magnetic particles are ideally suited for applications

involving high-throughput proteomic screening, small-

scale protein isolation, immunomagnetic isolations or

cell separation experiments. With magnetic beads,

affinity purification of tagged proteins, antigens,

antibodies and nucleic acids can be done conveniently

and quickly, with minimal time necessary for separation

of the solid-phase from solution. In addition, immobi-

lized substrates remain biologically active and can be

eluted in small volumes or serve as ligands in

subsequent pull-down or target interaction experiments

involving DNA or proteins.

Advantages:

* Convenient:

No centrifugation required; matrix can be regener-

ated without loss of binding capacity.

* Efficient:

Minimal sample loss during pipetting because

magnetic beads concentrate at the side of the tube

instead of the bottom.

Immunomagnetic Isolation

Protein A & Protein G Magnetic Beads are an affinity matrix for the small-scale

isolation and purification of immunoglobulins. A truncated form of recombinant

Protein A or G is covalently coupled to a nonporous paramagnetic particle & exhibits

high affinity for subclasses of IgG from many species including human, rabbit and

mouse. The linkage is stable and leak resistant over a wide pH range. This permits the

immunomagnetic purification of IgGs from ascites, serum or cell culture supernatants;

the matrix can then be regenerated without loss of binding capacity. These Beads can

be used to immunoprecipitate target proteins from crude cell lysates using selected

primary antibody. In addition, specific antibodies can be chemically cross-linked to the

Protein A or Protein G coated surface to create a reusable immunoprecipitation bead,

avoiding the co-elution of antibody with target antigen.

Immobilised Secondary Antibodies

Goat Anti-Rabbit and Goat Anti-Mouse IgG Magnetic Beads have the

antibody stably linked to nonporous paramagnetic beads. They are suitable for immu-

noassays that employ rabbit IgG polyclonal or mouse IgG monoclonal primary

antibodies. Cell separations and sorting can be accomplished using rabbit or mouse

antibody to defined cell surface antigens.

Applications:

* Magnetic cell sorting, cell separation & flow

cytometry

* Immunoprecipitation

* Immunoasssays

* Antibody immobilisation

* Small-scale purification of fusion proteins,

antibodies, immunoglobulins, biotinylated

proteins or mRNA, etc.

Cell population

Blood or Lysates

Labelled Primary

Antibody

NEB’s Immobilised

Secondary Antibody Bound to magnet.

Wash and detect

Magnetic Protein Purification

Chitin and Amylose Magnetic Beads are affinity matrices for the small-scale

purification of the relevant fusion proteins, after which the matrix can be regenerated

without loss of binding capacity; or, immobilised fusion proteins can be used in

subsequent experiments to capture target proteins from crude cell lysates that interact

with the immobilised fusion protein.

Anti-MBP Magnetic Beads permit the immunomagnetic isolation of MBP-fusion

proteins.

NEB’s Magnetic

Separation Rack

holds six 1.5 ml

tubes & features

strong neodym-

ium magnets for

fast separations.

Biotin-Labeled Isolation

Streptavidin Magnetic Beads are 1 µm superparamagnetic beads covalently

coupled to a highly pure form of streptavidin. Captured substrates can be useful as

ligands in subsequent experiments including mRNA isolation and the capture of

primary or secondary antibodies.

For current prices see www.genesearch.com.au/prices

Other Protein Tools

Glycosidases

Glycosidases are used to get information about the

carbohydrate groups attached to glycoproteins and

glycopeptides. Endoglycosidases cleave entire carbohy-

drate groups from proteins and exoglycosidases remove

monosaccharides from the nonreduced ends of the

carbohydrate. A reduced end is one generated by an

endoglycosidase. Researchers frequently use an endogly-

cosidase followed by one or more exoglycosidases and

then analyze the products using SDS-PAGE or liquid

chromatography.

Protein Kinases & Phosphatases

The reversible addition of phosphate groups to proteins is important for the transmis-

sion of signals within eukaryotic cells and, as a result, protein phosphorylation and

dephosphorylation regulate many diverse cellular processes. Most NEB protein kinases

& phosphatases are recombinant, and all are rigorously tested for contaminating

protease and phosphatase activity.

Kinases

Abl Protein Tyrosine Kinase (Abl)

Akt1/Protein Kinase B

Calmodulin-dependent Protein Kinase II

(CaMKII)

Casein Kinase I (CKI)

Casein Kinase II (CKII)

Cdc2 Protein Kinase

CDK2-cyclin A

cAMP-dependent Protein Kinase (PKA),

catalytic subunit

Glycogen Synthase Kinase 3 (GSK-3)

p42 MAP Kinase (Erk2)

Phosphatases & Inhibitors

LAR Protein Tyrosine Phosphatase (LAR)

YOP Protein Tyrosine Phosphatase (YOP)

T-Cell Protein Tyrosine Phosphatase (TC

PTP)

Lambda Protein Phosphatase (λ-PPase)

Protein Phosphatase 1 (PP1)

Protein Phosphatase Inhibitor 2 (I-2)

Protein Ser/Thr Phosphatase (PSP) Assay

System

Protein Tyr Phosphatase (PTP) Assay

System

Endoglycosidases

EndoH

EndoH f

PNGase F

Exoglycosidases

β-N-Acetyl-hexosaminidase

α-N-Acetyl-galactosaminidase

α1-2 Fucosidase

β1-3 Galactosidase

α1-2,3 Mannosidase

α1-6 Mannosidase

Neuraminidase

α2-3 Neuraminidase

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