EIF4E antibody (AA 121-217)

Catalog No. ABIN1386772

This independently validated Rabbit Polyclonal antibody specifically detects EIF4E in WB, IHC (p), IF (cc), IF (p) and IHC (fro). It exhibits reactivity toward Human.

Quick Overview for EIF4E antibody (AA 121-217) (ABIN1386772)

Target: EIF4E (Eukaryotic Translation Initiation Factor 4E (EIF4E))

Reactivity: Human

Host: Rabbit

Clonality: Polyclonal

Conjugate: This EIF4E antibody is un-conjugated

Application: Western Blotting (WB), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunofluorescence (Cultured Cells) (IF (cc)), Immunofluorescence (Paraffin-embedded Sections) (IF (p)), Immunohistochemistry (Frozen Sections) (IHC (fro))

Product Details anti-EIF4E Antibody

Binding Specificity: AA 121-217

Purpose: eIF4E Polyclonal Antibody

Cross-Reactivity: Human, Mouse, Rat

Purification: Purified by Protein A.

Immunogen: KLH conjugated synthetic peptide derived from human eIF4E

Isotype: IgG

Application Details

Application Notes: WB(1:300-5000),

Restrictions: For Research Use only

Independent Validation (IHC)

Validation #029640 (Immunohistochemistry)

by: Immunohistochemistry Core, NYU Langone

No.: #029640

Date: 03/14/2014

Lot Number: 140106

Method validated: Immunohistochemistry

Positive Control: Human pancreas

Negative Control: Human colon

Notes: Clear strong signal was detected in positive control sample. Note a minor amount of expected background staining in negative control sample.

Validation Images

Figure 1: Human pancreas tissue stained with anti-EIF4E (brown) and counterstained with hematoxylin.

Figure 2: Human colon tissue stained with anti-EIF4E (brown) and counterstained with hematoxylin.

Figure 3: Human pancreas tissue stained with isotype control antibody (brown) and counterstained with hematoxylin.

Figure 4: Human pancreas tissue stained with secondary antibody only (brown) and counterstained with hematoxylin.

Full Methods

Primary Antibody:

• Antigen: Eukaryotic Translation Initiation Factor 4E (EIF4E)
• Catalog number: ABIN1386772
• Lot number: 140106

Secondary Antibody:

• Antibody: Biotinylated goat anti-rabbit/anti-mouse (Kit)
• Lot number: D07640BA

Full Protocol:

• Immunohistochemistry was performed on a Ventana NEXes automated platform; instrument manufacturer specific reagents are italicized.
• 1. Slides were preheated in convection oven at 60°C for 30 min
• 2. Deparaffinization procedure: - 3 changes of Xylene, 5 min each - 3 changes of 100% Ethanol, 3 min each - 3 changes of 95% Ethanol, 3 min each - Rinsed in distilled water, 3 changes
• 3. Heat retrieval procedure - Slides retrieved in 10.0 mM Citrate, pH6.0 in a 1000W microwave oven (~100°C) for 15 min. - Slides were allowed to cool (in citrate) for 30 min. - Slides were washed x 3 in Distilled water
• 4. NEXes instrument procedure, iView DAB paraffin protocol (*abridged*): - Slide chamber warmed to 37°C
• 5. Slides rinsed with *reaction buffer* x3
• 6. *iView Inhibitor (H2O2)* applied and incubated for 4 min
• 7. Slides rinsed with *reaction buffer*
• 8. Antibody Application - Primary antibody diluted 1:250 in PBS (100 microliter applied/slide) - Ventana Isotype control applied neat - Slides Incubated overnight at room temperature (~12 hours ~25°C)
• 9. Slides rinsed with *reaction buffer* x3
• 10. *iView Biotinylated IgG* applied and incubated for 8 min
• 11. Slides rinsed with *reaction buffer*
• 14. *iView Streptavidin-Horseradish Peroxidase* applied and incubated for 8 min
• 15. Slides rinsed with *reaction buffer*
• 16. *iView DAB/H2O2* applied and incubated for 8 min
• 17. Slides rinsed with *reaction buffer*
• 18. *iView Copper* applied and incubated for 4 min
• 19. Slides rinsed with *reaction buffer*
• 20. Slides washed in Dawn Detergent/tap water
• 21. Counterstain Procedure - Hematoxylin (Leica 560 MX) 30 sec - Slides washed in tap water, 1 min - Decolorized (10% Acetic Acid in 70% ethanol), 1 min - Slides washed in tap water, 1 min - Bluing (Austin Clear Ammonia), 1 min - Slides washed in tap water, 1 min
• 22. Dehydration/coverslipping procedure: - 3 changes of 95% Ethanol, 3 min each - 3 changes of 100% Ethanol, 3 min each - 3 changes of Xylene, 5 min each - Mounted with Permount
• 23. Imaging: Leica SCN 400F Whole Slide Scanner with Digital Image Hub and Leica Slidepath software

Experimental Notes:

• Deviations from protocol/procedure supplied by manufacturer (attached).
• Step 1: Heated tissue 60°C for 30 minutes; manufacturer heats for 45 minutes.
• Step 2: No ethanol wash was performed during deparaffinization; manufacturer includes 1 wash of 80% ethanol for 3 minutes.
• Step 3.1: Slides were heated for 15 minutes; manufacturer provides a range of 15-20 minutes.
• Step 3.2: Slides were cooled for 30 minutes; manufacturer cools for 20 minutes.
• Step 4: Italicized reagents and incubation time are fixed instrument parameters.
• Step 5: Secondary species-specific serum block not used; manufacturer blocks with 5% normal goat serum for 2 hours.
• Step 8.1: Antibody diluted in PBS at 1:250; manufacture did not recommend diluent or dilution.
• Step 8.2.1: Primary antibody incubated at room temperature overnight; manufacturer incubates overnight 4°C with agitation.

Handling

Format: Liquid

Concentration: 1 μg/μL

Buffer: 0.01M TBS( pH 7.4) with 1 % BSA, 0.02 % Proclin300 and 50 % Glycerol.

Preservative: ProClin

Precaution of Use: This product contains ProClin: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: 4 °C,-20 °C

Storage Comment: Shipped at 4C. Store at -20C for one year. Avoid repeated freeze/thaw cycles.

Expiry Date: 12 months

Target Details for EIF4E

Target: EIF4E (Eukaryotic Translation Initiation Factor 4E (EIF4E))

Alternative Name: eIF4E

Background:

Synonyms: CBP, EIF4F, AUTS19, EIF4E1, EIF4EL1, Eukaryotic translation initiation factor 4E, eIF-4E, eIF4E, eIF-4F 25 kDa subunit, mRNA cap-binding protein

Background: Recognizes and binds the 7-methylguanosine-containing mRNA cap during an early step in the initiation of protein synthesis and facilitates ribosome binding by inducing the unwinding of the mRNAs secondary structures. Component of the CYFIP1-EIF4E-FMR1 complex which binds to the mRNA cap and mediates translational repression. In the CYFIP1-EIF4E-FMR1 complex this subunit mediates the binding to the mRNA cap.

Gene ID: 1977

UniProt: P06730

Pathways: BCR Signaling