Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [2]
- Immunocytochemistry [1]
Submit
Validation data
Reference
Comment
Report error
- Product number
- 700994 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- EGFR Recombinant Rabbit Monoclonal Antibody (30H45L42)
- Antibody type
- Monoclonal
- Antigen
- Other
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Antibody clone number
- 30H45L42
- Vial size
- 100 µg
- Concentration
- 0.5 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
No comments: Submit comment
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of EGFR (Fig a) was performed by loading 30 µg of A-431 Control (lane1), A-431 EGFR knockout (lane 2) whole cell extracts using Novex®NuPAGE®10 % Bis-Tris gel, XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. EGFR was detected at ~ 180 kDa using Recombinant Rabbit Monoclonal EGFR Antibody (Product # 700994, 1 µg/mL) at in 5% skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG (H+L) Recombinant Superclonal™ Secondary Antibody HRP conjugate (Product # A27036, 1:4000 dilution) was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106). Densitometric analysis of this Western blot is shown in histogram (Fig b). Loss of signal in CRISPR mediated knockout (KO) confirms that antibody is specific to EGFR.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), HEK-293 (Lane 2), T-47D (Lane 3), HCT116 (Lane 4), A-431 (Lane 5), A549 (Lane 6), U-87 MG (Lane 7) and H1975 (Lane 8). The blot was probed with Recombinant Rabbit Monoclonal EGFR Antibody (Product # 700994, 1 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Recombinant Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 180 kDa band corresponding to EGFR was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE® 10 % Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane using wet transfer system. The membrane was probed with the relevant primary and secondary antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of EGFR was performed using 90% confluent log phase A-431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with EGFR Antibody (30H45L42), Recombinant Rabbit Monoclonal (Product # 700994) at 5 microgram/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing membranous localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.