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Novel Affinity Tag System for Animal Host cells! - From Japan Next Generation Affinity Tag System version 2
PA tag


PA tag is a versatile protein tagging system using a super high affinity monoclonal antibody (NZ-1) against a peptide tag (12 amino acids).
Monoclonal antibody (NZ-1)has high affinity and high specificity against PA tag and is applicable to Immunocytochemistry (ICC), Western blotting and flow cytometry.
In addition, purification of PA tag fusion protein by IP and regeneration of beads are available.
PA tag system will be a unique and powerful tool for production in animal host cells.



  INDEX

  1. PA tag System
  2. PA tag Expression Vectors
  3. Anti PA tag, Rat Monoclonal Antibody (NZ-1)
  4. Affinity-purification from culture supernatant with PA tag
  5. Application: Western blotting
  6. PA tag Product Line-up
    ⇒ PA tag inserted Gene expression vectors
    ⇒ PA tag - for Detection and Purification of recombinant proteins
    ⇒ Related Products: Antibiotics
  7. Leaflet Link to Leaflet

  ⇒ LINK to





1. PA tag System





2. PA tag Expression Vectors



Vector Expression pattern Host MCS promoter
pCAG Transient expression Primate animal cells (human, monkey, dog)
Animal cells (mouse and rat)
CAG


We also offer a lineup of products of other vectors with antibiotic resistance genes different from the above (Please see the p.4 of this leaflet.). Detailed information on the sequencing primers and other vector sequences are included in the instruction manual supplied with the product. The sequence information (compressed file) and Multi Cloning Site (MCS) can be downloaded from our website.


3. Anti PA tag, Rat Monoclonal Antibody (NZ-1)

NZ-1 is a monoclonal antibody against PA tag (GVAMPGAEDDVV). NZ-1 can be used to detect recombinant PA tag fusion proteins expressed in animal cells and Escherichia coli. The antibody may provide some background signals in application to certain cell lines expressing human podoplanin at a high level, such as the human glioblastoma cell line LN319. The absence of cross reactivity has been confirmed in mouse-, rat-, hamster-, and dog-derived cell lines.
[Concentration]
  Indicated on the label.
  (Approximately 1.0 mg/mL)

[Formulation]
  1× PBS (pH 7.5) aqueous solution

[Clone No.]
  NZ-1

[Subclass]
  IgG2a

[Working Dilution]
  Western Blot 0.01-1 μg/mL
  Immunoprecipitation 10-50 μg/assay
  Flow Cytometory 0.1-10 μg/mL
  Immunocytochemistry 0.1-10 μg/mL

[Storage]
  Keep at 2-10°C.
  Avoid repeated freeze and thaw.

4. Affinity-purification from culture supernatant with PA tag


Data was provided by Junichi Takagi and Yuki Fujii, Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University
Protocol
Purification of protein of interest
 1) Transfection of mammalian cells (HEK293T)
   Transfect with cells at 40%–60% confluency.
   Culture medium: D-MEM containing 5% FBS
 2) Recovery of culture supernatant and absorption of proteins to resin
   Collect approximately 420 mL of the culture supernatant
   48-72 h after transfection. Add 8 mL of anti-PA tag
   antibody beads
(net 4 mL) and mix for 2 h (at 2–10°C).
 3) Resin recovery
   Recover beads from step 2 in a 50-mL column.
 4) Resin wash
   Wash beads five times with four beads volumes of TBS
   Buffer (20 mM Tris-HCl, 150 mM NaCl).


   5) Competitive elution using PA tag peptide
    Add one bead’s volume of PA tag peptide solution
    (final concentration: 0.1 mg/mL) in 1× TBS
    (pH 7.5) and collect the eluate in 10 portions to
    recover PA tag fusion-protein X.

   6) SDS-PAGE
    Sample volume:
    c. sup:5 μL, flow through: 5 μL, wash:5 μL,
    Peptide elution (1–10): 10 μL each
PA tag fusion-protein X was successfully purified, independently of the PA tag location.
→ Affinity-purification to a very high purity is possible!

5. Application: Western blotting

Recombinant isocitrate dehydrogenase (IDH1), tandemly fused with DYKDDDDK tag and PA tag at the C terminus, was expressed in osteosarcoma cells and detected with antibodies specified below, following SDS-PAGE of the cell lysate.

Data were provided by Drs. Yukinari Kato and Mika Kaneko, Regional Innovation Center, Tohoku University Graduate School of Medicine.

Lane 1 Primary Ab: Anti IDH1 Ab (RcMab-1), 1 µg/mL
       2nd Ab: Anti Rat IgG-HRP conjugated, 1000-fold dilution
 Lane 2 Primary Ab: Anti PA tag Ab, 1 µg/mL
       2nd Ab: Anti Rat IgG-HRP conjugated, 1000-fold dilution
 Lane 3 Primary Ab: Anti DYKDDDDK Ab, 3.5 µg/mL
       2nd Ab: Anti mouse IgG-HRP conjugated, 1000-fold dilution

Sample volume: 10 µg each
Exposure time:10 minutes

NZ-1 showed a high detection sensitivity
comparable with that of native antibodies.



6. PA tag Product Line-up

⇒ PA tag inserted Gene expression vectors

These products are gene expression vectors for mammalian cells. They enable expression of target proteins fused PA tag.

Vector pattern Host MCS promoter
pCAG Transient expression Primate animal cells (human, monkey, dogs)
Animal cells (mouse, rat)
CAG

Selection Marker Product Name Pkg. Size Wako Cat. No. Storage
Condition
E. coli Animal Cells
Bleomycin pCAG-Ble PA tag-C
⇒ Package insert       ⇒ Sequence
20 μg 161-26861 Keep at -20°C.
pCAG-Ble PA tag-N Signal Plus
⇒ Package insert       ⇒ Sequence
20 μg 168-26871
pCAG-Ble PA tag-N
⇒ Package insert       ⇒ Sequence
20 μg 161-27721
Blasticidin S pCAG-Bsd PA tag-C
⇒ Package insert       ⇒ Sequence
20 μg 165-26881
pCAG-Bsd PA tag-N Signal Plus
⇒ Package insert       ⇒ Sequence
20 μg 162-26891
pCAG-Bsd PA tag-N
⇒ Package insert       ⇒ Sequence
20 μg 168-27731
Hygromycin B pCAG-Hyg PA tag-C
⇒ Package insert       ⇒ Sequence
20 μg 165-26901
pCAG-Hyg PA tag-N Signal Plus
⇒ Package insert       ⇒ Sequence
20 μg 162-26911
pCAG-Hyg PA tag-N
⇒ Package insert       ⇒ Sequence
20 μg 164-27711
Kanamycin G418 pCAG-Neo PA tag-C
⇒ Package insert       ⇒ Sequence
20 μg 169-26921
pCAG-Neo PA tag-N Signal Plus
⇒ Package insert       ⇒ Sequence
20 μg 166-26931
pCAG-Neo PA tag-N
⇒ Package insert       ⇒ Sequence
20 μg 167-27701

Notice to purchaser: These products are for research use only. If you would like to resell them and/or develop new vectors constructed by DNA fragments and/or sequence information of the products, please contact us (cservice@wako-chem.co.jp).



⇒ PA tag - for Detection and Purification of recombinant proteins

Product Name Package Size Wako Cat. No. Storage Condition
Anti PA tag, Rat Monoclonal Antibody (NZ-1)
⇒ Package insert
200 μg 016-25861 Keep at 2–10°C.
1 mg 012-25863
Anti PA tag, Rat Monoclonal Antibody (NZ-1)
Peroxidase conjugated
200 μL 015-25951
1 mL 011-25953
Anti PA tag Antibody Beads 2 mL (Net 1 mL) 012-25841
10mL (Net 5 mL) 018-25843
50 mL (Net 25 mL) 016-25844
PA tag Peptide
⇒ Package insert
5 mg 167-25501 Keep at -20°C.
25mg 163-25503
PA tag Washing Solution 50 mL 169-27261 Keep at Room Temperature.


⇒ Related Products: Antibiotics

Product Name Pkg. Size Wako Cat. No. Storage Condition
Blasticidin S Hydrochloride 100 mg 029-18701 Keep at 2-10°C.
10 mg/mL Blasticidin S Hydrochloride, HEPES Solution 1 mL 026-18711 Keep at −20°C.
1mL x 10 022-18713
50 mg/mL G-418 Sulfate Solution, Animal-derived-free 20 mL 071-06431 Keep at 2-10°C.
100 mL 077-06433
G-418 Sulfate 1 g 078-05961
5 g 074-05963
25 g 076-05962


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