Genetic Research
Detection & Purification of Tagged Recombinant Protein
Anti Tag, Monoclonal
Antibodies
Anti Ta
T g, Monoclonal Antibodies
page
Working Dilution/assay
Western blot
IP
Description (( ): Clone No.)
Tag
Anti DYKDDDDK tag (1E6)
1:1,000 ∼ 1:10,000
2∼10 μg/assay
1:1,000 ∼ 1:20,000
-
-
10∼50 μL beads
slurry
1:1,000 ∼ 1:10,000
5∼10 μg/assay
1:4,000 ∼ 1:16,000
-
1:1,000 ∼ 1:10,000
2∼10 μg/assay
1:4,000 ∼ 1:16,000
-
1:500 ∼ 1:5,000
5∼10 μg/assay
1:500 ∼ 1:2,000
1∼10 μg/assay
-
10∼50 μL beads
slurry
-
1∼10 μg
1:500 ∼ 1:1,000
-
Anti GST (5A7) (200 μg (#013-21851))
1:500 ∼ 1:5,000
1∼10 μg
Anti GST (5A7), HRP (100 μL (#011-21891))
1:250 ∼ 1:1,000
-
Anti c-Myc (9E10) (200 μg (#017-21871))
1:500 ∼ 1:5,000
1∼10 μg/20 μL
Anti c-Myc (9E10), HRP (100 μL (#014-21901))
1:250 ∼ 1:2,000
-
-
10∼50 μL beads
slurry
Anti HA, mAb (4B2) (200 μg (#014-21881))
1:500 ∼ 1:5,000
1∼10 μg
Anti HA, mAb (4B2), HRP (100 μg (#011-21911))
1:250 ∼ 1:2,000
-
-
10∼50 μL beads
slurry
(200 μg (#018-22381); 1 mg (#014-22383); 5 mg (#012-22384))
2
DYKDDDDK
Anti DYKDDDDK tag (1E6), HRP
(100 μL (#015-22391))
Anti DYKDDDDK tag (1E6), Antibody Beads
7
(1 mL (#012-22781); 5 mL (#018-22783); 25 mL (#016-22784))
Anti 6 x His (9F2) (C-terminal specific)
(200 μg (#010-21861))
Anti 6 x His (9F2), HRP (C-terminal specific)
(100 μL (#013-23171))
(200 μg (#011-23091)
6 x His
Anti 6 x His (9C11), HRP (N･C-terminal)
(100 μL (#010-23181)
Anti 6 x His (21-48) (N･C-terminal)
(200 μg (#017-23211)
Anti 6 x His (28-75) (N･C-terminal)
(200 μg (#014-23221)
Anti 6 x His (48-75), Antibody Beads
7
(1 mL (#019-23391); 5 mL (#015-23393))
Anti GFP (mFX73) (for IP and Immunohistochemistry)
5
GFP
(100 μL (#012-20461); 100 μLx5 (#018-20463))
Anti GFP (mFX75) (for Western Blotting)
(200 μg (#012-22541))
6
GST
6
c-Myc
Anti c-Myc (9E10), Antibody Beads
7
(1 mL (#017-23071); 5 mL (#013-23073))
6
HA
Anti HA (4B2) Antibody Beads
7
Host
Synthesized
peptide
DYKDDDDK
Mouse
Isotype
IgG2b
-
IgG1
Anti 6 x His (9C11) (N･C-terminal)
3
Antigen
(1 mL (#014-23081); 5 mL (#010-23083))
Synthesized
peptide 6 x His
Mouse
IgG1･κ
IgG3･κ
-
GFP & GFP
partial peptide
Mouse
IgG2a･κ
GST
Mouse
IgG2b
Synthesized
peptide
EQKLISEEDL
Mouse
Synthesized
peptide
YPYDVPDYA
Mouse
IgG1
-
IgG2b
-
Tagged Proteins for Peptide Elution
page
Tag
DYKDDDDK
8
Description
DYKDDDDK Peptide (5 mg (#044-30951); 25 mg (#040-30953))
6 x His
6 x His Peptide
c-Myc
c-Myc Peptide (5 mg (#132-16361); 25 mg (#138-16363))
HA
(5 mg (#087-09251); 25 mg (#083-09253))
HA Peptide (5 mg (#088-09161); 25 mg (#084-09163))
Application
Note
Each Peptide is artificially
synthesized and used for the
competitive elution of tag fusion
proteins from anti tag antibody in
solution after immunoprecipitation.
Lyophilized; Content (HPLC)≧90%
Lyophilized; Content (HPLC)≧95%
Lyophilized; Content (HPLC)≧95%
Lyophilized; Content (HPLC)≧95%
Positive Control for Western Blotting
9
It is 74 kDa recombinant protein, consisted of 7 different tags
(DYKDDDDK, 6 x His, GFP, GST, c-Myc, HA and TRX) for detection
with each different antibody. It is used as a positive control for the
checking of epitope tag antibodies in western blot analysis.
Multitope, Denatured, recombinant,
Multitag Antigen Solution (10 ng/ μL)
(500 μL (#139-16371)
Solution;
Protein Conc.:
10 ng/ μL
Others
10
Purification
of 6 x His
fusion Protein
Cell Lysis Buffer
10 (for mammalian
cells)
Metal Chelate Agarose Set (Ni, Co, Cu, Zu)
(1 mL x 4 (#298-67401)
This kit is used to immobilize protein such as an antibody by chemically binding to
p-Nitrophenyloxycarbonyl bond of silica beads surface.
RIPA (Radio-Immunoprecipitation Assay) Buffer RIPA Buffer is used to prepare the cell lysate from adherent
(100 mL (#182-02451); 259 mL (#188-02453)
and suspension cultured mammalian cells.
Cell Lysis Buffer M (1 kit (#290-69301)
Eespecially recommended to use for the extraction of
cytoplasmic proteins.
Ready to Use Solution
Antibody Immobilization Kit
11
Antibody
Immobilization
Antibody Immobilization Kit IP
(1 kit (#290-69301)
This kit is used to immobilize protein such as an antibody by chemically
binding to p-Nitrophenyloxycarbonyl bond of silica beads surface.
Various applicabion at RIP
assay are available.
Anti Tag, Monoclonal Antibodies
01
High Affinity DYKDDDDK Antibody
H
Anti DYKDDDDK tag, Monoclonal Antibody (Clone No. 1E6)
Anti DYKDDDDK tag, Monoclonal Antibody, Peroxidase conjugated
High Affinity with DYKDDDDK sequence & Good cost performance
Product information
● Concentration : 0.5 mg/mL
● Subclass : IgG2b
● Composition : 50 v/v% Glycerol
1 x PBS solution
● Antigen : DYKDDDDK peptide
conjugated with KLH.
● Clone No. : 1E6
● Storage
: -20℃
Anti DYKDDDDK tag, Monoclonal Antibody
（Wako Cat. # 018-22381, 200μg）
Immunoprecipitation (IP)
Western blot analysis (WB)
1
2
3
4
IP
Silver staining
150
Lane1: Wako 1E6 Dilution ratio 1 : 1,000
Lane2: Wako 1E6 Dilution ratio 1 : 10,000
Lane3: Vender S Dilution ratio 1 : 1,000
Lane4: Vender S Dilution ratio 1 : 10,000
100
75
M 1 2 3 4
WB
1 2 3 4
Lane1: Input
(COS7 cell lysate including
DYKDDDDK tag fusion protein)
Lane2: Mouse IgG IP fraction
Lane3: Wako 1E6 IP fraction
Lane4: Vender S IP fraction
150
100
75
50
50
IgG Ｈ chain
37
37
25
Detection performance
Wako 1E6 antobody ＞ Vender S antibody
20
Western blot analysis for the detection of DYKDDDDK tag fusion protein.
Cell line: COS7
Dilution ratio of primary antibody: 1: 1,000, and 1: 10,000
WB
（kDa）
1
2
3
1
2
DYKDDDDK tag fusion
protein A
72
55
20
Detection performance
Wako 1E6 antibody ≒ Vender S antibody
〔Experimental condition〕
3
110
25
Western blot analysis for the detection of DYKDDDDK tag fusion protein.
Wako 1E6
Vender S Antibody
〔Experimental condition〕
Cell line: COS7
Beads matrix: Protein G immobilized magnetic beads
antigen: 1x105 cells / assay
immobilized antibody: 5 μg / 20 μL of 10% slurry
IgG L chain each
Elution: 20 μL of 2% SDS sample buffer / assay
Applied IP sample: 10 μL / lane
Dilution ratio of primary antibody for WB: 1:1,000
(Vender S antibody)
Lane1: Mock HEK293 cells
Lane2: Clone 1
Lane3: Clone 2
Cell line: HEK293
Selection: The transfected cells were randomly
selected after 24 hours from plasmid transfection.
Cell lysis buffer: RIPA buffer
Dilution ratio of primary antibody: 1:10,000
Recognition performance of DYKDDDDK peptide
sequence Wako 1E6 antibody ＞ Vender S antibody
Wako DYKDDDDK (1E6) -HRP
Antibody Conc.
Data was provided by:
Dr. Shuichi Kusano (Associate Professor) Division Persistent and
Oncogenic Viruses Kagoshima University, Center for Chronic Viral
Diseases
1
0.5mg/mL
2
3
0.25mg/mL
2
3
Vender S Antibody-HRP
Antibody Conc.
1
0.5mg/mL
2
3
0.25mg/mL
2
3
High recognition performance
of DYKDDDDK sequence !!
Anti-FLAG M2
Wako 1E6
2 4 6 8
2 4 6 8
Loaded（μL）
02
Data was provided by: Dr. Taisuke Tomita (Associate Professor).
Graduate School of Pharmaceutical Sciences University of Tokyo.
Wako 1E6 antibody ≒ Vender S antibody
02
Data was provided by: Dr. Yutaka Terada. Cancer Research Institute of Kanazawa Univ. (Japan)
Lane 1: 293T Cell Lysate
Lane 2: 293T Cell Lysate from transient cells
expressing FLAG fusion protein A
Lane 3: 293T Cell Lysate from transient cells
expressing FLAG fusion protein B
Blocking：1 x TBS-T including 5% Skim Milk. 4℃, O/N
Antigen-Antibody reaction：4℃, 120min
Detection：LAS-3000, Expose time 15min.
Wako 1E6 antibody ＞ Vender S antibody
Code No.
Package size
Description
018-22381
200 μg
014-22383
1 mg
Anti DYKDDDDK tag, Monoclonal Antibody（1E6）
5 mg
012-22384
015-22391
Anti DYKDDDDK tag, Monoclonal Antibody, Peroxidase Conjugated（1E6）
100 μL
1 mL
012-22781
018-22783
5 mL
Anti DYKDDDDK tag Antibody Beads（1E6）
25 mL
016-22784
044-30951
5 mg
DYKDDDDK Peptide
040-30953
25 mg
Anti
A
tti 6 x Hi
His, Monoclonal Antibody
Variety of Highly Sensitive 6 x His Monoclonal Antibodies are available!
Wako established 4 kinds of clones which produce antibodies with different characteristics.
You can choose an appropriate one by the selection guide as follows:
6 x His Monoclonal Antibodies Selection Guide
N- 6 x His Recombinant Protein -C
Clone #
Recognition
Western Blot
Immunoprecipitation
ELISA
Working Dilution
N-terminal
C-terminal
Ab Conc.
N-terminal
C-terminal
1:1,000 ∼ 1:10,000
ND
+++++
5∼10 μg/assay
ND
++
1:4,000 ∼ 1:16,000
ND
+++++
1:1,000 ∼ 1:10,000
+++++
++++
2∼10 μg/assay
+++++
++
1:4,000 ∼ 1:16,000
+++++
++++
21-48
1:500 ∼ 1:5,000
+++
++++
5∼10 μg/assay
+++
+++
28-75
1:500 ∼ 1:2,000
++
++
1∼10 μg/assay
+++++
+++++
Anti 6 x His (9F2)
(200 μg (#010-21861))
N- Recombinant Protein 6 x His -C
9F2
C-teminal
Anti 6 x His (9F2), HRP
(100 μL (#013-23171))
Anti 6 x His (9C11)
(200 μg (#011-23091)
9C11
Anti 6 x His (9C11), HRP
(100 μL (#010-23181)
N･C-terminal
Anti 6 x His (21-48)
(200 μg (#017-23211)
Anti 6 x His (28-75)
(200 μg (#014-23221)
ND: Non Detection:
The data listed in the table above are obtained from our in-house invetigation and do not show the common characteristics of all the 6 x His fusion
proteins. Please be advised that depending on the characteristics of the intended antigen-antibody reactions may be affected, leading to different
results from the guide above.
FAQ for Clone Selection Guide
Q1 : Which clones are best suited for detection of N-terminal 6 x His?
A1 : 9C11 (WB), 9C11 (IP), 28-75 (IP)
Q2 : Which clones are best suited for detection of C-terminal 6 x His?
A2 : 9F2 (WB), 28-75 (IP)
Q3 : Which clones can recognize N･C-terminal 6 x His and are best suited for WB?
A3 : 9C11 (WB)
Q4 : Which clones can recognize N･C-terminal 6 x His and best suited for IP?
A4 : 28-75 (IP)
Q5 : Which clone is multipurpose and can be used in WB and IP?
A5 : 21-48 (WB･IP)
Q6 : Which clone is the highest sensitive in WB?
A6 : 9C11 (WB)
Q7 : Which clone is the highest sensitive in IP?
A7 : 28-75 (IP)
Q8 : Which is the most inexpensive clone that recognizes C-terminal 6 x His?
A8 : 9F2 (Inexpensive! (C-terminal))
Q9 : Which is the most inexpensive clone that recognizes N-terminal 6 x His?
A9 : 21-48 (Inexpensive! (N-terminal))
Anti Tag, Monoclonal Antibodies
03
Application data
Prices
● WB
9C11
28-75
28
-7
5
21
-4
8
9F
2
inexpensive
9C
11
9F
2
21
-4
8
28
-7
5
C- terminal 6 x His
（ His.
Ve H8
nd
er
M）
250
150
9C
11
（kDa）
（ His.
Ve H8
nd
er
M）
N- terminal 6 x His
21-48
9F2
6 x His-LacZ
（116kDa）
100
75
50
37
p35-6 x His
25
20
①SDS-PAGE
Gel ：10-20% SuperSep, 20mA
Sample：N terminal His
（293T expressing 6 x His LacZ (1×105 cell)）
C terminal His
（60ng p35-6 x His+ CHO Lysate(1×10 5 cell)）
②WB
Primary Antibody ：1μg/mL
Secondary Antibody：anti-mouse IgG 1:5000
Detection ：ImmunoStar LD
15
10
（kDa）
250
150
100
75
Anti 6 x His, MAb(9F2) (Wako Cat#. 010-21861)
50
Dilution ratio: 1:5,000
whole cell lysate
Antigen: 10 μL of the
including 6 x His fusion protein.
37
25
20
15
10
250
250
150
8
5
-4
-7
（kDa）
C- terminal 6 x His
10
%
In
pu
（
Ve
t
nd
9C er M
11 ）
9F
2
21
-4
8
28
-7
5
10
%
（
I
Ve npu
nd t
e
9C r M）
11
9F
2
-4
8
28
-7
5
N- terminal 6 x His
21
1%
I
（ npu
t
Ve
nd
9C er M
11 ）
9F
2
21
-4
8
28
-7
5
pu
（
t
Ve
nd
9C er M
11 ）
9F
2
In
1%
（kDa）
WB
C- terminal 6 x His
21
Silver staining
N- terminal 6 x His
28
● IP
150
6 x His-LacZ
（116kDa）
100
75
6 x His-LacZ
（116kDa）
100
75
50
50
37
37
p35-6 x His
IP matrix：magnetic beads
Antigen quantity：
N terminal His: 293T expressing
6 x His LacZ
(1×105 cell)
C terminal His: 60ng p35-6 x His+
CHO Lysate
(1×106 cell)
p35-6 x His
Antibody ：5μg/Assay
Detection：＜Silver staining＞
quantity
＜WB＞
Primary Antibody :
anti-His (His.H8) 1mg/mL
Secondary Antibody :
anti-mouse IgG 1:5,000
Detection：ImmunoStar LD
25
25
20
20
15
15
10
10
Anti 6 x His, MAb(9F2) (Wako Cat#. 010-21861)
IP
（kDa） 1
2
3
IP
4
5
6
7
〔Experimental condition〕
Cell:
293T
Selection: The transfected 293T cells were randomly selected after
24 hours after the transfection of plasmids which were
harboring genes of fusion proteins.
72→
55→
43→
34→
Vender N mAb 2.5 μg/mL
Lane 1: 6 x His fusion protein Input
Lane 2: Flow through fraction
after IP
Lane 3: Elution fraction after IP
Lane 4: M. W. Marker
Vender Q His
Wako His（9F2）
2 4 6 8
2 4 6 8
Loaded（μL）
Wako 6 x His mAb 2.5 μg/mL
Lane 5: 6 x His fusion protein Input
Lane 6: Flow through fraction
after IP
Lane 7: Elution fraction after IP
Data was provided by Dr. Taisuke Tomita (Associate Professor). Graduate School
of Pharmaceutical Sciences University of Tokyo.
Wako 9F2 antibody ＞ Vender Q antibody
04
（ mFX75 ）
Anti GFP, Monoclonal Antibody（ mFX73）
A
Western blot analysis（WB）
（Clone No. mFX75）
Wako
mFX75
mFX75 clone for WB
（kDa） C 1 2
250
150
100
75
Product information
● Concentration：Approx. 1mg/mL
(Indicated on label)
Vernder M Vender S
C 1 2
C 1 2
Vender N
C 1 2
50
● Composition ：D-PBS(-), 10mmol/l Hepes,
pH7.4, 10mmol/l Sodium Azide.
37
● Clone No. ：mFX75
GFP
25
20
● Subclass ：IgG2a･κ
● Antigen ：GFP and the partial peptide of GFP.
15
10
● Storage ：-20℃
Comparison of detection performance for WB
Dilution ratio of primary antibody：1：500 ∼ 1：1,000
C ：HepG2 cell Lysate
（5×104cells）
Lane 1：HepG2 cell Lysate（5×104cells）+rGFP 1ng
Lane 2：HepG2 cell Lysate（5×104cells）+rGFP 10ng
Immunoprecipitation(IP), Immunohistochemistry(IHC) (Clone No. mFX73)
mFX73 clone for IP & IHC
Product information
● Concentration：Approx. 1mg/mL
(Indicated on label)
● Composition ：D-PBS(-), 10mmol/l
Hepes, pH7.4, 10mmol/l
Sodium Azide.
（kDa）
Anti-GFP
250
150
100
75
BSA
50
DAPI
IgG H chain
37
● Clone No. ：mFX73
25
● Subclass ：IgG2a･κ
20
● Antigen ：GFP and the partial
peptide of GFP.
M 1 2 3 4
GFP
IgG L chain
Anti-serotonin
15
● Storage ：-20℃
Working concentration
IP ：1 ∼ 5 μg / 5 μl Sepharose
IHC ：0.5 ∼ 1 μg / mL
Lane 1：GFP
Lane 2：Elution fraction (Wako mFX73 immobilized beads)
Lane 3：Elution fraction (Vender M mAb immobilized beads)
Lane 4：Elution fraction (Vender B mAb immobilized beads)
〔Experimental condition〕
Cell lysate：HepG2
Beads matrix：Protein G immobilized sepharose
Amounts of antigen：0.5 μg of GFP /assay
Elution：SDS sample buffer
Applied IP sample onto gels：10 μl/lane
Detection：Silver staining
※Cross-reactivity
Positive：wild-type GFP, GFP variants(A. victoria)
Negative：TurboGFP（Pontellina plumata ）, Phi-YFP（Phialidium sp.）,KFP-Red
（Anemonia sulcata）
Code No.
012-20461
018-20463
012-22541
Description
Anti Green Fluorescent Protein, Monoclonal Antibody (mFX73)
Anti Green Fluorescent Protein, Monoclonal Antibody (mFX75)
C. elegans strain：unc-18
（e81）
；unc-18::EGFP L4
Fixation condition：4% Paraformaldehyde /1%
Glutaraldehyde, 4℃, O/N.
Primary antibody：Anti GFP Antibody（mFX73）
1 μg/mL ＋ Anti Serotonin serum（1:500）
Secondary antibody：Cy3 labelled Anti-mouse
antibody
（1:200）＋Biotin labelled Anti rabbit IgG
（1:200） /ABC complex（1:100）
Detection：DAB/fluorescent
Wako’
s anti GFP antibody(mFX73) was
interacted with the serotonin positive neurons,
neuron cell body and process on the head of
C. elegans.
（Data：Department of Physiology, Tokyo Women’
s
Medical University Cshool of medicine. Dr. Shohei
Mitani）
Package size
100 μL
100 μL × 5
200 μg
Anti Tag, Monoclonal Antibodies
05
Anti GST, c-Myc, HA, Monoclonal Antibody
A
Western blot analysis (WB)
1 Anti GST, MAb (Wako Cat#. 013-21851)
Dilution ratio: 1:1,000, antigen: 10 μL of the 1/10 diluted
cell lysate including GST fusion protein.
〈GST〉
whole
〈c-Myc〉
（kDa） 2
（kDa） 1
250
150
100
75
50
2 Anti c-Myc, MAb (Wako Cat#. 017-21871)
Dilution ratio: 1: 4,000, antigen: 10 ng of c-Myc fusion protein.
3 Anti HA, MAb (Wako Cat#. 014-21881)
25
20
4 Anti GST, MAb, Peroxidase Conjugated
(Wako Cat#. 011-21891)
15
Dilution ratio: 1:1,000, antigen: 10 μL of the 1/10 diluted
cell lysate including GST fusion protein.
250
150
100
75
50
37
37
25
20
25
20
（kDa） 4
250
150
100
75
50
(Wako Cat#. 014-21901)
Dilution ratio: 1: 1,000, antigen: 10 ng of c-Myc fusion protein.
6 Anti HA, MAb, Peroxidase Conjugated
15
15
10
10
3
10
whole
5 Anti c-Myc, MAb, Peroxidase Conjugated
（kDa）
250
150
100
75
50
37
Dilution ratio: 1: 2,000, antigen: 5 ng of HA fusion protein.
〈HA〉
(Wako Cat#. 011-21911)
Dilution ratio: 1: 500, antigen: 10 ng of HA fusion protein.
（kDa） 5
250
150
100
75
50
（kDa） 6
250
150
100
75
50
37
37
37
25
20
25
20
25
20
15
15
15
10
10
10
Immunoprecipitation (IP)
〔Experimental condition〕
Cell:
293T
Selection: The transfected 293T cells were randomly selected after
24 hours after the transfection of plasmids which were
harboring genes of fusion proteins.
1 Anti GST, MAb (Wako Cat#. 013-21851)
2
3
4
5
55 →
43 →
34 →
Lane 1:
293T whole cell lysate
Lane 2:
Wako GST mAb 1 μg/mL
Lane 3:
Wako GST mAb 2 μg/mL
Lane 4:
Vender S mAb 2 μg/mL
Lane 5: Mouse IgG
3 Anti HA, MAb (Wako Cat#. 014-21881)
IP
（kDa） 1
55 →
43 →
34 →
06
2
3
2 Anti c-Myc, MAb (Wako Cat#. 017-21871)
IP
IP
（kDa） 1
Beads matrix: Protein A/G immobilized magnetic beads
Detection:
Western blot
Dilution ratio of primary antibody: 1：1,000
4
5
Lane 1: 293T whole cell
lysate
Lane 2: Wako HA mAb
1 μg/mL
Lane 3: Wako HA mAb
2 μg/mL
Lane 4: Vender R mAb
2 μg/mL
Lane 5: Mouse IgG
（kDa） 1
34 →
26 →
17 →
2
3
4
5
Lane 1: 293T whole cell
lysate
Lane 2: Wako c-Myc mAb
1 μg/mL
Lane 3: Wako c-Myc mAb
2 μg/mL
Lane 4: Vender S mAb
2 μg/mL
Lane 5: Mouse IgG
High Affinity & High Quality！
H
！
Anti DYKDDDDK tag Antibody Beads
Anti 6 x His Antibody Beads
Anti c-Myc Antibody Beads
Anti HA Antibody Beads
A series of High quality Affinity Beads for recognition of DYKDDDDK tag,
HA tag, c-Myc tag and 6 x His tag
［Tag Antibody Beads］
DYKDDDDK
Description
012-22781（ 1 mL）
018-22783（ 5 mL）
016-22784（25 mL）
Wako Cat. No.（Package size）
019-23391（1 mL）
015-23393（5 mL）
Antibody quantity
Antibody clone No.
Antibody Subclass
Antibody binding capacity/1ml of beads
2.0 mg/mL
1E6
IgG1
48-75
IgG3・κ
9E10
approx. 1.0 mg
approx. 1.0∼2.0 mg
approx. 0.9 mg
-20℃
Purification of DYKDDDDK fusion protein
nd
W er S
ak
o
Ve
S
o
ak
er
approx. 1.5 mg
-20℃
2∼10℃
Peptide Elution Outline
Elution methods
SDS
DYK
nd
4B2
IgG1
1.8∼2.1mL slurry/mL resin
Storage
W
8.5 mg/mL
7.5 mg/mL
Setting Volume
Ve
1 x PBS (pH 7.4), 50% glycerol,
0.02 w/v% sodium azide.
4% Agarose
Beads matrix
250
150
HA
017-23071（1 mL） 014-23081（1 mL）
013-23073（5 mL） 010-23083（5 mL）
1 x TBS (pH 7.4),
1 x PBS (pH 7.4), 50% glycerol,
0.02 w/v% sodium azide.
0.05 w/v% sodium azide.
Formulation
（kDa） M
c-Myc
6ｘHis
：Tag fusion proteins
：Endogenous host proteins
M
〈Elution Methods〉
DYK: Competitive elution by
DYKDDDDK peptide
SDS: Denaturing elution by 2% SDS
100
75
Crude cell lysate
Anti Tag Antibody
Beads
Immunoprecipitation
＜Non-specific background＞
Wako 1E6 beads＜Vender S beads
50
37
＜Recovery of DYKDDDDK fusion protein＞
Wako 1E6 beads＜Vender S beads
Competitive elution
Tag Peptide
25
20
DYKDDDDK tagged
rProtein (19 kDa)
15
Purification performance
Wako 1E6 beads ＞ Vender S beads
〔Experimental condition〕
〈Cell〉
E. coli
〈Beads〉
20 μL of Anti DYKDDDDK tag Antibody Beads(Wako) (/assay)
20 μL of Vender S Affinity Beads(/assay)
〈Amounts of antigen solution〉
20mg of E. coli whole cell lysate including DYKDDDDK
fusion protein(/assay)
〈Antigen-Antibody reaction〉
4℃, 3 hours
Peptide Elution
● Applicable to Antigen elution
under neutral conditions (pH 7.0)
● Simple condition setting
● Highly efficient purification
Purified fusion protein
〈Elution〉
20 μL of 150 μg/mL DYKDDDDK peptide (#044-30951) (/assay)
20 μL of 8 x SDS sample buffer(/assay)
→ 4℃, 30 minutes
〈Applied IP sample〉10 μL/lane
〈Detection〉
Silver staining
Anti Tag, Monoclonal Antibodies
07
Purification of 6 x His fusion protein
（kDa）
1 2 3
（kDa）
250
150
100
75
250
150
100
75
50
37
25
1 2 3
1 2 3
In
pu
t
SDS sample
buffer
%
In
pu
t
Acidic
buffer
%
1 2 3
WB（anti-6 x His MAb）
SDS sample
buffer
10
Acidic
buffer
10
IP（Silver staning）
50
IgG Heavy chain
IgG Heavy chain
37
p35-His
p35-His
25
IgG Light chain
IgG Light chain
20
15
20
15
Lane 1：Vendor S Anti polyhistidine Agarose Beads
Lane 2：Wako 1 mg / Beads
Lane 3：Wako 2 mg / Beads
Lane 1：Sigma Monoclonal Anti polyHistidine Agarose
Lane 2：Wako 1 mg / Beads
Lane 3：Wako 2 mg / Beads
Highly Purified Tagged Proteine for Peptide Elution
H
DYKDDDDK Peptide, HA Peptide, c-Myc Peptide,
and 6 x His Peptide
These tagged peptides are used for the competitive elution of Tag fusion proteins
from antigen-antibody complex in solution after immunoprecipitation.
●
●
●
●
●
Content（HPLC）: ≧95%
Amino Acid Sequences: DYKDDDDK, HHHHHH(6 x His), YPYDVPDYA(HA), EQKLISEEDL(c-Myc)
Molecular weight check by MULDI-TOF MS
Appearance: Lyophilized powder（TFA salt form）
Storage: Keep at -20℃
【Working Concentration】
Standard: 100 ~ 150 μg/mL in 1 x TBS
Please consider the optimum condition for peptide elution.
【Preparation of stock solution】
Dissolve in 1 x TBS (10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L Sodium Chloride) to a final concentration
of 5 mg/mL.
Aliquot and store at -20 ℃. Do not repeated freeze and thaw.
Description
08
Package Size (Wako Catalog No.)
DYKDDDDK Peptide
5 mg (#044-30951); 25 mg (#040-30953)
HA Peptide
5 mg (#088-09161); 25 mg (#084-09163)
c-Myc Peptide
5 mg (#132-16361); 25 mg (#138-16363)
Positive Control of Western blot
P
Multitope, Denatured
Multitope is 74 kDa recombinant protein which was consisted of seven different tags for
detection with each different antibodies. Multitope protein is used as a positive control
for the checking of performance of epitope tag antibodies in western blot analysis.
Features
Epitopes
1. TRX (Thiodedoxin)
2. 6 x His (internal & C-terminal : HHHHHH)
3. GFP (Green fluorescent protein)
4. HA (Hemagglutinin : YPYDVPDYA)
5. DYKDDDDK
6. GST (Glutathione S-transferase)
7. c-Myc (EQKLISEEDL)
● Positive control for WB
● Ready-to-Use denatured solution
for SDS-APGE
● Single band
Multitope primary structure
H2N
TRX
6 x His
GFP
HA
Flag
GST
Mye 6 x His
COOH
Multitope Application
● SDS-PAGE
● WesternBlot
His
1 2
M 1
170
130
95
72
M: M.W. marker
1 : Multitope 20ng
GFP
1 2
1
HA
2
Flag
1 2
GST
1 2
Myc
1 2
C-His
1 2
170
1：Multitope 20ng
2：Multitope 50ng
130
95
55
43
72
34
26
55
43
17
34
＜Primary Antibodies＞ His：6 x His (9C11) (Wako Cat.#011-23091) 1: 5000, GFP: Anti GFP (mFX75) (Wako Cat.#012-22541) 1:1000, HA: Anti HA
(4B2) MoAb (Wako Cat.#014-21881) 1: 1000, DYKDDDDK: Anti DYKDDDDK (1E6) (Wako Cat.#018-22381) 1: 5000, GST:
Anti GST (5A7) (Wako Cat.#013-21851) 1: 1000, c-Myc: Anti c-Myc (9E10) (Wako Cat.#017-21871) 1:1000, C-His: Anti 6
x His(9F2) (Wako Cat.#010-21861) 1: 1000
＜Secondary Antibody＞ Goat anti Mouse IgG-HRP 1: 5000
＜Detection＞ Chemi-luminescent detection system
●
●
●
●
●
●
Application ：SDS-PAGE followed by Western Blot
Formulation ： 50 mmol/L Tris-HCl (pH6.8), 2 w/v% SDS, 10v/v% Glycerol, 0.1 w/v% bromophenol blue, 1.5 w/v% DTT.
Molecular weight ：Applox. 74 kDa
Protein concentration：10 ng/μL
Storage ：-20℃. Do not freeze and thaw cycles.
Precaution ：Component of storage buffer will be precipitated by storage at -20℃. In this case, please warm to room
temperature and mixed gently. We recommend that you confirm whether precipitant was completely dissolved.
Description
Multitope, Denatured, recombinant, Solution（10 ng/μL ）
Package Size (Wako Cat. No.)
500 μL (#139-16371)
Anti Tag, Monoclonal Antibodies
09
Metal Chelate Agarose (Purification of 6 x His fusion proteins)
M
These products can be used for purification of 6 x His tagged recombinant
protein by immobilized metal ion affinity chromatography (IMAC).
● Beads matrix: Crosslink 6% Agarose
● Ligand : Iminodiacetic Acid
● Beads size : 40 ∼ 180 μm
● Composition : 20 v/v% Ethanol
(50% suspension)
Binding capacity
Metal ion
Ni
20 ∼ 40 μmol/mL gel
2+
20 ∼ 40 μmol/mL gel
Co2+
2+
Cu
5 ∼ 20 μmol/mL gel
Zn2+
5 ∼ 20 μmol/mL gel
Metal Chelate Agarose Set (Ni, Co, Cu, Zn)
(Wako Cat.#298-67401)
Comparison of recovery efficiency of each agaroses
（kDa） 1
2
3
4
5
6
7
8
9 10
97
66
45
FIPA-6 x His
30
20.1
14.4
Purification of F1PA (fuculose-1-phosphate aldolase)
-6 x His fusion protein from
cell lysate by each
metal chelate agarose.
Lane
Lane
Lane
Lane
Lane
Lane
Code No.
298-67401
145-07981
141-07983
149-07984
263-01871
038-19791 031-19781 Lane 7 : Wako
Lane 8 : Vender
Lane 9 : Wako
Lane 10: Vender
1 : M. W. Marker
2 : F1PA-6 x His fusion protein
3 : Wako
Cu-Agarose
4 : Vender
5 : Wako
Ni-Agarose
6 : Vender
Description
Metal Chelate Agarose Set (Ni, Co, Cu, Zn)
Ni-Agarose
Zn-Agarose Cu-Agarose
Co-Agarose Zn-Agarose
Co-Agarose
Package size
1 mL each
5 mL
10 mL
100 mL
5 mL
5 mL
5 mL
Cell Lysis Buffer ( for mammalian cells)
C
These lysis buffer are used for preparation of cell lysate from mammalian cells. The prepared cell lysate were used
for several biological applications, such as immunoprecipitation, western blot and reporter assay. These sterilized
lysis buffer are supplied as a ready to use solution.
RIPA Buffer
RIPA (Radio-Immunoprecipitation Assay) Buffer is used to prepare the cell lysate from adherent and suspension cultured mammalian cells.
〔Formulation〕 50mmol/L Tris-HCl, pH8.0, 150 mmol/L Sodium Chloride, 0.5 w/v% Sodium Deoxycholate, 0.1 w/v% sodium
Dodecyl Sulfate, 1.0 w/v% NP-40 substitute
Cell Lysis Buffer M
The lytic potential is lower than RIPA Buffer, therefore this product is used for preparation of cell lysate in mild condition.
It is especially recommended to use for the extraction of cytoplasmic proteins.
〔Formulation〕 20 mmol/L Tris-HCl, pH7.4, 200 mmol/L Sodium Chloride, 2.5 mmol/L Magnesium Chloride,
0.05 w/v%NP-40 substitute
Description
Code No.
182-02451
188-02453
038-21141
034-21143
RIPA Buffer
Cell Lysis Buffer M
Package size
100 mL
250 mL
100 mL
250 mL
Related Products
10
160-19501
Protease Inhibitor Mixture, DMSO Solution. for Mammalian Cell and
Tissue Extracts
1 Vial
167-19511
Protease Inhibitors Mixture, for Protease and Esterase
1 Vial
167-24381
Phosphatase Inhibitor Cocktail SolutionⅠ (×100)
1 mL
160-24371
Phosphatase Inhibitor Cocktail Solution Ⅱ (×100)
1 mL
IP（Immunoprecipitation）＆ RIP（RNA Immunoprecipitation）Assay
Antibody & Protein Immobilization Beads
Antibody Immobilization Kit IP
[Active group] -nitrophenyloxycarbonyl（PNP）
(Wako Catalog No.290-69301)
This kit is to immobilize proteins including antibodies on carrier beads.
When used in the immunoprecipitation, the kit is applicable to a
variety of antibody species because immobilization by proteins such
as Protein G, which is a common technique, is not performed. Since
Antibody Immobilization Beads contained in this kit are also used as a
kit content in Wako microRNA Isolation kit series, these are
field-proven in the RIP Assay.
Features
●Applicable to RIP
(RNA Immunoprecipitation) Assay
Kit contents（1 kit）
●Usable a Desktop Centrifugation (6,000 rpm) for IP
● Antibody Immobilization Beads
● Antibody Immobilization Buffer
●Easy Decantation after Centrifugation
● Blocking Buffer
●Low Non-Specific Binding to Protein
and Nucleic Acids
● Washing Buffer (Neutral)
●Applicable to Immobilization of various species
of antibodies
● Storage Buffer
● Washing Buffer (Acidic)
200 mg × 1 bottle
10 mL × 1 bottle
10 mL × 1 bottle
80 mL × 1 bottle
20 mL × 1 bottle
20 mL × 1 bottle
The purpose of this kit is antibody immobilization.
●Applicable to Immobilization of Proteins (Avidin, Protein G, etc.)
Therefore this kit does not include any cell lysis reagents or antigen elution
reagents for immunoprecipitation (IP) experiments.
Low Background !!
Ｍ
HEK293
THP-1
FM3A
1 2 3
1 2 3
1 2 3
＜Specificity test＞
SCC131
Vender P Agarose beads
Vender G Sepharose beads
Wako Beads
1 2 3
（kDa）
250
150
100
↓
Immobilization of Anti Human Albumin MoAb.
↓
75
IgG H
50
chain
Mix with each cell lysates（HEK293, THP-1,
FM3A, SCC131）
（1×10 6 cells）. 2∼10℃, 3 hours.
↓
35
IgG L
25
chain
Wash × 3 times
↓←0.1M Glycine-HCl (pH2.5)
Elution of non-specific proteins.
↓
SDS-PAGE of the eluted solution and silver staining
20
Lane1 : Eluted solution (Vender P Agarose beads) 1/2 vol.
Lane2 : Eluted solution (Vender G Sepharose beads) 1/2 vol.
Lane3 : Eluted solution (Wako Beads) 1/2 vol.（Protocol 2.※1）
※1：Protocol to immobilize antibody with the affinity beads, which were
Low background
&
Strong binding capacity of antibody
washed by acidic buffer(shown on the package insert)
Anti Tag, Monoclonal Antibodies
11
Batch method can be carried our even when using a desktop centrifuge.
Broad interface beween gel and solution
Vender
G
Wako
6,000rpm
30sec
Beads slurry
A desktop centrifuge
Vender G
Vender
G
Wako
Wako
ビーズ遠心分離後
After centrifugation
Wako
Sharp interface beween gel and solution
Applications
1. IP of Human Albumin（Recovery test）
（kDa）
M 1 2 3 4
250
150
100
75
Human Albumin
IgG H chain
50
37
Procedures
1 x 10 6 HeLa cell lysate ＋ 0.2 μg Human
Albumin
↓←2.0mg Anti Human Albumin PoAb (10μL)
immobilized beads
↓ ↓
Immunoprecipitation. 2 ∼ 10℃, 3 hours.
↓ ←Washing Buffer
Wash x 3 times
↓ ←Elution Buffer
Eluted Solution
M:
ONE Point
Beads SEM figure
M.W. Marker
10μm
Lane1: Input (0.2μg Human Albumin) 1/2 vol.
Lane2: Input (1 x 10 6 HeLa cell lysate) 1/100 vol.
Lane3: Wako beads
（Anti Human Albumin MoAb Beads）
（Washing Buffer (Neutral)）
Eluted Solution ½ vol.
Lane4: Wako beads
（Anti Human Albumin MoAb Beads）
（Washing Buffer (Acidic)） Eluted Solution ½ vol.
IgG L chain
25
15
■ Core Matrix : Silica Beads
■ Average Beads size : 5μm
RIP Assay
2. RIP Assay
microRNA purification from human cell line
（nt）
M
1
2
3
4
500
200
100
5
LaneM : M.W.Marker
Lane 1 : Synthesized single
strand RNA
（22nt）1ng
Lane 2 : HeLa
Lane 3 : HepG2 human
Lane 4 : HEK293
Lane 5 : P388D1 mouse
50
microRNA
20
RIP Assay is RNA immunoprecipitation
method by using anti RNA-binding protein
monoclonal antibody. RIP Assay is used
by RNA researchers for the investigation of
mRNA, siRNA, microRNA and Non-coding
RNA which are bound with RNA-binding
proteins.
Recently, life science researches are
focusing on RIP Assay of Argonaute
proteins (Ago1, 2, 3, 4) for microRNA and
siRNA functional research, especially
identification of target mRNA.
In the near future, RIP Assay of Ago
proteins always will be used for the
development of nucleic acid pharmaceuticals
and investigation of unknown microRNAs.
Immunoprecipitation
Affinity Beads
Anti RNA binding
Proteins Antibody
RNA
RNA binding
Proteins
RNA Purification
RNA
Microarray, Real-Time qPCR,
Cloning, Sequence
After the making of Anti Human Ago2 MoAb immobilized beads by Antibody Immobilization
kit, the purified microRNA fraction from human cultured cell lines(HeLa, HepG2, HEK293)
were specifically detected by Urea-PAGE. Cell number of each cell line is approximately
5×106/Lane.
Description
Package Size (Wako Cat. No.)
Storage
Antibody Immobilization Kit Wako
1 kit (#290-69301)
Keep at 2∼10℃
The running costs are dependent on the quantity of beads used for IP. In the case of using 2.0 mg beads per IP, the content of this kit is equivalent to 100 IPs.
・Listed products are intended for laboratory research use only, and not to be used for drug, food or human use. Please visit our online catalog to search for
other products from Wako ; www.e-reagent.com. This brochure may contain products that cannot be exported to your country due to regulations.
Bulk quote requests for some products are welcomed. Please contact us.
www.wako-chem.co.jp
11
01
1153.0IBF
Anti Tag, MAb
EASY Handling !!