Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [1]
- Immunocytochemistry [1]
Submit
Validation data
Reference
Comment
Report error
- Product number
- PA5-17712 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-SHC (Tyr239, Tyr240) Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- It is not recommended to aliquot this antibody.
- Reactivity
- Human, Mouse, Rat
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Storage
- -20°C
No comments: Submit comment
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of A-431 (Lane 1), A-431 treated with EGF (200 ng/mL for 10 minutes) (Lane 2), A-431 treated with Afatinib followed by EGF (0.5 uM for 6 hours, 200 ng/mL for 10 minutes) (Lane 3), A549 (Lane 4) and A549 treated with EGF (200 ng/mL for 10 minutes) (Lane 5). The blot was probed with Anti-Phospho-SHC (Tyr239, Tyr240) Rabbit Polyclonal Antibody (Product # PA5-17712, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 52 kDa band corresponding to Phospho-SHC (Tyr239, Tyr240) along with 66 kDa SHC isoform was detected and observed to increase upon EGF treatment across the cell lines tested. Pre-treatment with EGFR-antagonist, Afatinib, resulted in inhibition of Phospho-EGFR in A-431 cell line. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of Phospho-SHC (Tyr239,Tyr240) 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 15 minutes and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-SHC (Tyr239,Tyr240) Rabbit Polyclonal Antibody (Product # PA5-17712) at 1:100 dilution in 0.1% BSA and incubated overnight at 4 degree Celsius and then labelled 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, 1:100). Panel d represents the merged image showing membranous localization. Panel e represents cells treated with antagonist, Afatinib (1uM for 6hrs) followed by EGF (200 ng/mL for 10 minutes), showing no signal. 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.