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ImmunocytochemistryAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Immunocytochemistry [2]
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- Product number
- 702338 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-EGFR (Tyr992) Recombinant Rabbit Monoclonal Antibody (3H24L5)
- Antibody type
- Monoclonal
- Antigen
- Synthetic peptide
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Antibody clone number
- 3H24L5
- 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.
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- For immunofluorescence analysis A431 cells were fixed and permeabilized for detection of endogenous EGFR (pY992) using the EGFR (pY992) Recombinant Rabbit Monoclonal Antibody (Product # 702338, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Detection and localization of EGFR (pY992) (green) in the membrane can be clearly observed in cells treated with EGF (100 ng/mL, 15 min) as compared to untreated cells. Antibody specificity was demonstrated by competition with the EGFR (pY992) peptide, which results in loss of signal. No competition was observed with the non-phospho peptide. The images were captured at 60X magnification.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of Phospho-EGFR (Tyr992) was performed using 90% confluent log phase A-431 cells treated with 200 ng/mL of EGF for 10 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-EGFR (Tyr992) Antibody (3H24L5), Recombinant Rabbit Monoclonal (Product # 702338) at 2 µg/mL in 0.1% BSA and overnight at 4 degree Celsius 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 cells treated with antagonist, Afatinib (1µM for 6hrs) followed by EGF (200 ng/mL for 10 minutes), showing reduced Phospho-EGFR staining. Panel f shows untreated cells with no signal. Panel g represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
