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This is a tool for efficient introduction of IgG antibodies into living cells using HVJ-E envelope (HVJ-E) vector for the purpose of analysis of the functions of cells and intracellular proteins.

GenomONE™ - CAb EX Antibody Delivery Reagent-Application


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CAb-2 Application

GenomONE-CAb EX dataPDF

1.Types of antibodies to which this kit is applicable
Delivery of various IgGs into HS-68 cells iHuman foreskin fibroblast; ATCC CRL-1635j

Species IgG subtype*1 Incorporation efficiency of antibody into HVJ-E*2 Introduction efficiency of antibody into HS-68 cells*3
Mouse pAb  ¢
IgG1  
IgG2a › ›
IgG2b  ¢
IgG3 › ¢
Rat pAb ¢ ¢
IgG1  ›
IgG2a ¢ ¢
IgG2b › ›
IgG2c ¢ ¢
Human IgG1  ›
IgG2  ›
IgG3 ¢ ¢
IgG4  ›
Rabbit pAb › ›
Goat pAb ¢ ¢

*1: pAb: polyclonal antibody, others: monoclonal antibody (control IgG)
*2: Incorporation efficiency of antibody into HVJ-EF
  ; more than 40%, ›; 20-40%, ¢; less than 20%
*3: Introduction of antibody into HS-68 cellsF
  ; efficiently delivered,@›;@can be delivered, ¢;@possible, but efficiency is low

 

2. Examples of cells into which IgG antibody can be introduced with this kit

Origin Cell Line Cell Type Antibody Type
Cancer, Adherent
Human HeLa S3 cervical epithelioid carcinoma mouse lgG pAB
mouse lgG 1mAB
Human A549 lung carcinoma mouse lgG 1mAB
Human SAS tongue carcinoma mouse lgG pAB
Human HT1080 fibrosarcoma mouse lgG pAB
Human MCF-7 mammary carcinoma mouse lgG2a mAB
Mouse P19 embryonal carcinoma mouse lgG pAB
Mouse Raw264.7 leukemic monocyte mouse lgG pAB
Cancer, Non- adherent
Human Jurkat T cell leukemia mouse lgG1 mAB
Mouse P3-X63-Ag8.653 myeloma mouse lgG1 mAB
Nomal, Adheren1
Human WI-38 lung fibroblast mouse lgG1 mAB
Human Hs68 skin fibroblast mouse lgG1 mAB
Mouse 3T3-L1 embryo fibroblast undifferentiated mouse lgG1 mAB
Mouse 3T3-L1, adipocyte embryo fibroblast differentiated mouse lgG1 mAB
Mouse BNL CL2 embryo hepatocyte mouse IgG1 mAb
Hamster BHK-21 kidney fibroblast mouse lgG1 mAB
Nomal, Primary cultured
Human HAEC aortic endothelial mmouse lgG1 mAB

pAb: polyclonal antibody, mAb: monoclonal antibody

 

3. Example of antibody introduction into living cells

3-1. Intracellular delivery of anti-NPC monoclonal antibody

yProcedurez
Cells, 1×104 cells/well@10%FCS/DME (8 well-chambered coverglass; Nunc 155411)
↓delivery of Anti-NPC mAb (2.5μg) into cells using GenomONE- CAb
↓incubation for 2 hr at 37°C
↓wash the cells(PBS)
↓fix the cells (4% PFA; for 15min, r.t.→PBS wash→0.2% Triton X-100; for 5min, r.t. →PBS wash→1%BSA/PBS; for 10min, r.t.
↓Alexa Fluor 488-Goat Anti-Mouse IgG, F(ab’)2 Fragment@(Invitrogen A11017)
↓wash the cells(PBS)
↓observation by confocal laser scanning microscopy

yAntibody delivered into living cellsz@ Monoclonal Anti-Nuclear Pore Complex Proteins, Clone 414, Mouse IgG1(SIGMA, N8786)

 

3-2. Introduction of anti-NPC antibody into various cell lines


Raw264.7
(ATCC-TIB-71)

Hs68
(ATCC-CRL1635)
Actin filaments were stained with phalloidin (red)

HeLa S3
(ATCC-CCL 2.2)

yProcedurez
Cells, 1×104 cells/well@10%FCS/DME (8 well-chambered coverglass ; Nunc 155411)
↓delivery of Anti-NPC mAb (2.5μg) into cells using GenomONE- CAb
↓incubation for 2 hr at 37°C
↓wash the cells (PBS)
↓fix the cells (4% PFA; for 15min, r.t.→PBS wash→0.2% Triton X-100; for 5min, r.t.→PBS wash→1%BSA/PBS; for 10min, r.t)
↓Alexa Fluor 488-Goat Anti-Mouse IgG, F(ab’)2 Fragment@(Invitrogen A11017)
↓wash the cells (PBS)
↓observation by confocal laser scanning microscopy

yAntibody delivered into living cellsz
Monoclonal Anti-Nuclear Pore Complex Proteins, Clone 414, Mouse IgG1 (SIGMA, N8786)

 

3-3. Introduction of anti-NPC antibody into 3T3-L1 cells

yProcedurez
Seed 3T3-L1 mouse embryo fibroblast (ATCC-CL-173),@1×104cells/well/8-well chambered coverglass, 10%FCS-DMEM
↓ two days after seeding (confluent), add MDI (0.5mM Methylisobutylxanthine, 1μM Dexamethasone, 10μg/mL Insulin)
↓ two days after differentiation; medium change to 10%FCS-DMEM ({Insulin), change the medium every two days
↓ seven days after differentiation; delivery of mAb (2.5μg) into cells using GenomONE- CAb
↓ incubation for 4hr at 37°C
↓ fix the cells (4% PFA; for 15 min, r.t. → 0.2% Triton X-100; for 5 min, r.t.
↓ Alexa Fluor 488-Goat Anti-Mouse IgG, F(ab’)2 <Invitrogen A11017>, 2μg/mL ×200μ‚k, incubation for 1hr at r.t.
↓ wash the cells(PBS) observation by confocal laser scanning microscopy

yAntibody delivered into living cellsz
Monoclonal Anti-Nuclear Pore Complex Proteins, Clone 414, Mouse IgG1(SIGMA, N8786)

 

3-4. Introduction of anti-α-tubulin antibody into Hs68 cells

yProcedurez
Hs68:@Human foreskin fibroblast iATCC-CRL-1635j, 1×104 cells/well, 0.5%FCS/DMEM(8 well-chambered coverglass ; Nunc 155411)
↓delivery of Anti-α-tubulin mAb (5μg) into cells using GenomONE- CAb
↓incubation for 2hr at 37°C
↓wash the cells (PBS)
↓fix the cells (4% PFA; for 15min at r.t., 0.2% Triton X-100; for 5 min at r.t., 1%BSA; o/n)
↓Alexa Fluor 488-Goat Anti-Mouse IgG, F(ab’)2 <Invitrogen A11017>
↓wash the cells (PBS)
↓observation by confocal laser scanning microscopy

yAntibodyz
Monoclonal Anti-α-tubulin, Clone DM1A, Mouse IgG1 (SIGMA, T6199)

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