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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).
A)
B)
kDa
1 2 3 4
kDa
1 2
3
4
83
62
48
66
56
43
33
37
25
27
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
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
(C) which contains only Carrier Protein 39
(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.
A)
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
B)
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
1
2
3
4
5
6
7
8
3.33
1.11
0.37
0.12
3.33
1.11
0.37
CP39-HA
kDa
83
62
48
HA
CP39-peptide
+ Abl Kinase
33
0.12
25
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
N-terminal C-terminal
Target Gene
Target Gene
T7 Promoter
MCS Intein Tag
Intein Tag
MCS
T7 Promoter
Target
Protein
Intein
Tag
Intein
Tag
Target
Protein
Load &
Wash
Chitin
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
90
80
70
60
50
40
30
20
10
0
2857.02
1449.43
1482.42
2317.02
10 µg each $114
ACTH (18-39)
2204.43
100
90
80
1650.49
2547.79
70
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)
60
1694.58
3340.65
50
1004.0 1532.8 2061.6 2590.4 3119.2 3648.0
Neurotensin
40
Mass (m/z)
ACTH (7-38)
30
Peaks from myoglobin:
Peaks from GluC:
M/z theoretical residues M/z theoretical residues
868.86 869.9 *
20
19-27 1650.49 1649.89
42-54
10
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
90
1782.73
80
1197.17
70
60
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
50
2323.40
40
30
1654.63
1031.91
20
10
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
H
– 27.0
H
Target Protein
Intein-tag
O
Cys
– 20.0
Step 2: Thiol Mediated
Cleavage
3 2 2
SO -CH -CH -SH
H
2
N
– 14.3
– 6.5
– 2.4
Target Protein
S
Intein-tag
O
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
-
2
SH
Bio-P1
– 83
– 62
Cys
O
Step 4: S-N Shift
NH
2
– 47.5
Target Protein
S
Bio-P1
O
Cys
Bio-P1 (Lyophilized)
For labeling proteins with biotin.
#P6607S
– 32.5
SH
H
– 25
Target Protein
N
Bio-P1
0.29 mg $197
Cys
O
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.
0
0
-
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
Plik z chomika:
morcys1
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