GenomONE™ - CAb EX Antibody Delivery Reagent-Application

GenomONE™ – CAb _EX top

GenomONE-CAb _EX data

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

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

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

pAb: polyclonal antibody, mAb: monoclonal antibody

3. Example of antibody introduction into living cells

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

【Procedure】
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

【Antibody delivered into living cells】　 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)

【Procedure】
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

【Antibody delivered into living cells】
Monoclonal Anti-Nuclear Pore Complex Proteins, Clone 414, Mouse IgG1 (SIGMA, N8786)

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

【Procedure】
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 ×200μＬ, incubation for 1hr at r.t.
↓ wash the cells(PBS) observation by confocal laser scanning microscopy

【Antibody delivered into living cells】
Monoclonal Anti-Nuclear Pore Complex Proteins, Clone 414, Mouse IgG1(SIGMA, N8786)

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

【Procedure】
Hs68:　Human foreskin fibroblast （ATCC-CRL-1635）, 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

【Antibody】
Monoclonal Anti-α-tubulin, Clone DM1A, Mouse IgG1 (SIGMA, T6199)