Antibodies to eukaryotic Initiation Factors (eIF) - FocusOn 094

Selected Antibody Panel to eIF Protein Family

Acris Antibodies offers a range of monoclonal and polyclonal antibodies to nearly all members of the eIF protein family. This collection is complemented by Abnova monoclonal antibodies distributed by Acris Antibodies within Europe and the USA. These antibodies are thoroughly evaluated in Western blotting and immunohistochemical staining methods (see’ Product list’).

Introduction

Eukaryotic initiation factors (eIF) are proteins involved in the initiation phase of eukaryotic translation. They function in forming a complex with the 40S ribosomal subunit and Met-tRNAi called the 43S preinitiation complex (PIC), recognizing the 5’ cap structure of mRNA and recruiting the 43S PIC to mRNA, promoting ribosomal scanning of mRNA and regulating recognition of the AUG initiation codon, and joining of the 60S ribosomal subunit to create the 80S ribosome. There exist many more eukaryotic initiation factors than prokaryotic initiation factors due to the greater biological complexity of eukaryotic cells.

eIF1, eIF2, eIF3

eIF1, eIF1A, and eIF3 all bind to the ribosome subunit-mRNA complex. They have been implicated in preventing the large ribosomal subunit from binding the small subunit before it is ready to commence elongation.

Eukaryotic Initiation Factor 2 (eIF2) is a protein required in the initiation of translation. In this fundamental process of life, the ribosome builds proteins according to the information encoded on the mRNA. eIF2 mediates the binding of tRNAmet to the ribosome in a GTP-dependent manner. eIF2 is a heterotrimer consisting of an alpha (also called eIF2S1), a beta (eIF2S2), and a gamma (eIF2S3) subunit. Once the initiation is completed, eIF2 is released from the ribosome bound to GDP as an inactive binary complex. To participate in another round of translation initiation, this GDP must be exchanged for GTP.

eIF2 is an essential factor for protein synthesis that forms a ternary complex (TC) with GTP and the initiator Met-tRNA. After its formation, the TC binds the 40S ribosomal subunit to form the 43S preinitiation complex (PIC). PIC-assembly is believed to be stimulated by the initiation factors eIF1, eIF2A and the eIF3 complex according to in vitro experiments. The 43S PIC then binds mRNA that has previously been unwound by the eIF4s. The 43S PIC and the eIF4 proteins form a new 48S complex on the mRNA which starts searching along the mRNA for the start codon (AUG). Upon base pairing of the AUG-codon with the Met-tRNA, eIF5 (which is a GTPase activating protein) is recruited to the complex and induces eIF2 to hydrolyse its GTP. This causes eIF2-GDP to be released from this 48S complex, and translation commences after recruitment of the 60S ribosomal subunit and formation of the 80S initiation complex. Finally, with the help of the guanine nucleotide exchange factor eIF2B, the GDP in eIF2 is exchanged for a GTP and the ternary complex reforms for a new round of translation initiation.

eIF2 activity is regulated by a mechanism involving both guanine nucleotide exchange and phosphorylation. Phosphorylation takes place at the α-subunit which is a target for a number of serine kinases that phosphorylate serine 51. Those kinases act as a result of stress such as amino acid deprivation (GCN2), ER stress (PERK), the presence of dsRNA (PKR) or Hemoglobin deficiency (HRI). Once phosphorylated, eIF2 shows increased affinity for its guanine nucleotide exchange factor eIF2B. However, eIF2B is only able to exchange GDP for GTP if eIF2 is in its unphosphorylated state. Phosphorylated eIF2, due to its stronger binding acts as an inhibitor of its own GEF (eIF2B). Since the cellular concentration of eIF2B is much lower than that of eIF2, even a small amount of phosphorylated eIF2 can completely abolish eIF2B activity by sequestration. Without the GEF, eIF2 can no longer be returned to its active (GTP-bound) state. Consequently translation comes to a halt since initiation is no longer possible without any available ternary complex.

Fig. 1: Western Blot using Acris eIF2S2 antibody Cat.-No. AP22365PU-N at 1:1000 dilution: Sample (30 µg whole cell lysate) 10% SDS PAGE. A: Hela B: A431 C: H1299 D: Hela
Fig. 2: Immunofluorescence analysis of paraformaldehyde-fixed A431, using Acris eIF2B5 antibody Cat.-No. AP22366PU-N (1:200; right figure merged with DNA probe)
Fig. 3: Staining of formalin-fixed, paraffin-embedded (FFPE) human kidney using Acris eIF2A antibody Cat.-No. AP08404PU-N at 1:50 dilution, followed by biotinylated secondary antibody, alkaline phosphatase-streptavidin and chromogen
Fig. 4: IHC analysis of mouse brain tissue (FFPE) with Acris eIF2B2 antibody Cat.-No. AP17313PU-N, followed by peroxidase conjugated secondary antibody and DAB staining

In mammals, eIF3 is the largest scaffolding initiation factor, made up of 13 subunits (a-m). It is roughly ~750 kDa, and it controls the assembly of the 40S ribosomal subunit on mRNAs that have a 5’ cap or an IRES (internal ribosomal entry site). eIF3 uses the eIF4F complex or IRES from viruses to position the mRNA strand near the exit site of the 40S ribosome subunit, thus promoting the assembly of the pre-initiation complex.

Fig. 5: Immunofluorescence analysis of methanol-fixed A431, using Acris eIF3A antibody Cat.-No. AP22367PU-N(1:200; right figure is co-stained with Hoechst)
Fig. 6: Staining of formalin-fixed, paraffin-embedded (FFPE) human breast sections using Acris eIF3B antibody Cat.-No. AP08406PU-N at 1:50, followed by biotinylated secondary antibody, alkaline phosphatase-streptavidin and chromogen
Fig. 7: Human breast carcinoma tissue (FFPE) reacted with Acris eIF3J antibody (N-term) Cat.-No. AP13969PU-N; peroxidase-conjugated secondary antibody / DAB staining
Fig. 8: Immunohistochemical analysis of paraffin-embedded Cal27 xenograft using Acris eIF3K antibody Cat.-No. AP22368PU-N at 1:100 dilution

eIF4

The eIF4 initiation factors include eIF4A, eIF4B, eIF4E, and eIF4G. eIF4F is often used to refer to the complex of eIF4A, eIF4E, and eIF4G.

eIF4 is responsible for the binding of capped mRNA to the 40S ribosomal subunit via eIF3. The mRNA cap is bound by eIF4E (25 kDa). eIF4G (185 kDa) acts as a scaffold for the complex whilst the ATP-dependent RNA helicase eIF4A (46 kDa) processes the secondary structure of the mRNA 5’ UTR to render it more conducive to ribosomal binding and subsequent translation. Together these three proteins are referred to as eIF4F.

For maximal activity eIF4A also requires eIF4B (80 kDa), which itself is enhanced by eIF4H (25 kDa). A study into wheat germ indicates that eIF4A has a higher binding affinity for ADP than ATP except in the presence of eIF4B, which increased the ATP binding affinity tenfold without affecting ADP affinity. Once bound to the 5’ cap of mRNA, this 48S complex then searches for the AUG start codon and translation begins.

Fig. 9: Staining of human lung carcinoma tissue (FFPE) using Acris eIF4A1 antibody Cat.-No. AP13970PU-N; peroxidase-conjugated secondary antibody / DAB staining
Fig. 10: Immunohistochemistry using monoclonal Novus antibody to eIF4A3 Cat.-No. H00009775-M01 on human salivary gland (FFPE) [antibody concentration 1 µg/ml]
Fig. 11: Staining of human breast carcinoma sections (FFPE) using Acris eIF4B antibody Cat.-No. AP11771PU-N, followed by peroxidase-conjugated secondary antibody and DAB staining
Fig. 12: Staining of human heart sections (FFPE) using Acris eIF4E antibody Cat.-No. AP08407PU-N (1:50), followed by biotinylated secondary antibody, alkaline phosphatase-streptavidin and chromogen
Fig. 13: Staining of human breast carcinoma tissue (FFPE) with Acris eIF4G1 antibody Cat.-No. AP14537PU-N using peroxidase-conjugated secondary antibody and DAB staining
Fig. 14: Human brain, cortex sections (FFPE) stained with Acris eIF4G2 antibody Cat.-No. AM08365PU-N at 10 µg/ml, followed by biotinylated secondary antibody, alkaline phosphatase-streptavidin and chromogen

eIF5

eIF5A is a GTPase-activating protein, which helps the large ribosomal subunit associate with the small subunit. It is required for GTP-hydrolysis by eIF2 and contains the unusual amino acid hypusine. eIF5B is a GTPase, and is involved in assembly of the full ribosome (which requires GTP hydrolysis).

eIF6

eIF6, also called integrin beta 4 binding protein (ITGB4BP), binds to the 60S ribosomal subunit and prevents its association with the 40S riboso-mal subunit to form the 80S initiation complex in the cytoplasm. It may behave as a stimulatory translation initiation factor of downstream insulin/growth factors. eIF6 is also involved in ribosome biogenesis, associates with pre-60S subunits in the nucleus and is involved in its nuclear export.

Key references

 
Zoll WL, Horton LE, Komar AA, Hensold JO, Merrick WC: Characterization of mammalian eIF2A and identification of the yeast homolog.
J. Biol Chem, Oct 2002; 277(40): 37079-87.
 
Telemann AA, Chen YW, Cohen SM: 4E-BP functions as a metabolic brake used under stress conditions but not during normal growth.
Genes Dev, Aug 2005; 19(16): 1844-8.
 
Clement PM, Henderson CA, Jenkins ZA, Smit-McBride Z, Wolff EC, Hershey JW, Park MH, Johansson HE: Identification and characterization of eukaryotic initiation factor 5A-2.
Eur J Biochem, Nov 2003; 270(21): 4254-63.

Further readings

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Primary Antibodies

Catalog No. Host Iso. Clone Pres. React. Applications  

EIF1AY antibody

Western Blot analysis of EIF1AY expression in transfected 293T cell line by EIF1AY monoclonal antibody (M01), clone 1B4.

Lane 1: EIF1AY transfected lysate(16.4 KDa).
Lane 2: Non-transfected lysate. Mouse IgG2a 1B4 Purified Hu E, WB
0.1 mg / €370.00
  Abnova Taiwan Corp.

EIF1 / SUI1 antibody

EIF1 / SUI1 Mouse IgM 2E1 Ascites Hu E, WB
0.2 ml / €370.00
  Abnova Taiwan Corp.

EIF2A (C-term) antibody

AP08404PU-N EIF2A antibody staining of Formalin-Fixed Paraffin-Embedded Human Kidney at 1/50 followed by biotinylated Goat anti-Rabbit IgG secondary antibody, Alkaline Phosphatase-Streptavidin and Chromogen. Rabbit IgG Aff - Purified Hu, Ms P, WB
50 µl / €470.00
  Acris Antibodies GmbH

EIF2A / EIF2S1 pSer51 antibody

Immunohistochemical analysis of Paraffin-Embedded Human breast carcinoma tissue using AP02522PU eIF2A antibody pSer51 Rabbit Aff - Purified Hu, Ms, Rt ICC/IF, P, WB
0.1 ml / €320.00
  Acris Antibodies GmbH

EIF2A / EIF2S1 pSer51 antibody

Immunohistochemical analysis of Paraffin-Embedded Human breast carcinoma tissue using AP02522PU eIF2A antibody pSer51 Rabbit Aff - Purified Hu, Ms, Rt ICC/IF, P, WB
50 µl / €240.00
  Acris Antibodies GmbH

EIF2A / EIF2S1 pSer51 antibody

Placenta: Formalin-Fixed Paraffin-Embedded (FFPE) Rabbit IgG Aff - Purified Hu, Ms, Rt P, WB
50 µg / €470.00
  Acris Antibodies GmbH

EIF2A / EIF2S1 pSer49 antibody

Figure 1. Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using eIF2a pSer49) Antibody (#AP08033PU). Rabbit Aff - Purified Hu, Ms, Rt ICC/IF, P, WB
0.1 ml / €320.00
  Acris Antibodies GmbH

EIF2A / EIF2S1 pSer49 antibody

Figure 1. Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using eIF2a pSer49) Antibody (#AP08033PU). Rabbit Aff - Purified Hu, Ms, Rt ICC/IF, P, WB
50 µl / €240.00
  Acris Antibodies GmbH

EIF2B1 / EIF2BA antibody

EIF2B1 MaxPab polyclonal antibody. Western Blot analysis of EIF2B1 expression in human colon. Mouse Purified Hu E(detection), WB
50 µg / €370.00
  Abnova Taiwan Corp.

EIF2B3 (C-term) antibody

Western blot analysis using AP11734PU-N EIF2B3 antibody in A375 cell line lysate. EIF2B3 (arrow) was detected using the purified antibody. Rabbit Ig Purified Hu WB
0.4 ml / €370.00
  Acris Antibodies GmbH

EIF2G / EIF2S3 antibody

Western Blot analysis of EIF2S3 expression in transfected 293T cell line by EIF2S3 monoclonal antibody (M01), clone 2C9.

Lane 1: EIF2S3 transfected lysate(51.1 KDa).
Lane 2: Non-transfected lysate. Mouse IgG2a 2C9 Purified Hu E, WB
0.1 mg / €370.00
  Abnova Taiwan Corp.

EIF3C / EIF3S8 antibody

Western Blot analysis of EIF3C expression in transfected 293T cell line (H00008663-T01) by EIF3C MaxPab polyclonal antibody.

Lane 1: EIF3S8 transfected lysate(100.43 KDa).
Lane 2: Non-transfected lysate. Mouse Serum Hu, Ms E(detection), WB
50 µl / €370.00
  Abnova Taiwan Corp.

EIF3D / EIF3S7 antibody

Immunohistochemistry (IHC) analyzes of eIF3D antibody (Cat.-No.: AP21173PU-N) staining in Paraffin-Embedded Human breast carcinoma tissue. Rabbit Aff - Purified Hu, Ms P, WB
0.1 mg / €350.00
  Acris Antibodies GmbH

EIF3E / EIF3S6 antibody

Western blot using affinity purified anti-eIF3S6/Int6 antibody shows detection of endogenous eIF3S6/Int6. Specific staining is not present in lysates containing lentiviral knockdown vectors (shRNA #1 and #2. Control vectors, specifically a scrambled sequence (Ctl NS) and a sequence against an unrelated gene (Ctl #2), were also used. Personal communication, J.Lee, NCI, Bethesda, MD. Rabbit IgG Aff - Purified Bov, Can, Hu, Mky, Ms, Rt E, WB
25 µl / €190.00
  Acris Antibodies GmbH

EIF3E / EIF3S6 antibody

EIF3S6 polyclonal antibody (A01), Lot # 060111JC01 Western Blot analysis of EIF3S6 expression in Y-79 ( Cat # L042V1 ). Mouse Serum Hu E, WB
50 µl / €290.00
  Abnova Taiwan Corp.

EIF3F / EIF3S5 (114-125) antibody

Figure 1. AP07581PU-N EIF3F antibody staining of Formalin-Fixed Paraffin-Embedded Human Brain, cerebellum at 2.5 µg/ml followed by biotinylated Goat anti-Rabbit IgG secondary antibody, Alkaline Phosphatase-Streptavidin and Chromogen. Rabbit Aff - Purified Bov, Chk, Hu, Ms, Rt E, ICC/IF, IP, P, WB
50 µg / €470.00
  Acris Antibodies GmbH

EIF3F / EIF3S5 antibody

Immunofluorescence of purified MaxPab antibody to EIF3S5 on HeLa cell. [antibody concentration 10 ug/ml] Mouse Purified Hu E(detection), ICC/IF, WB
50 µg / €370.00
  Abnova Taiwan Corp.

EIF3G / EIF3S4 antibody

Western Blot analysis of EIF3G expression in transfected 293T cell line (H00008666-T01) by EIF3G MaxPab polyclonal antibody.

Lane 1: EIF3S4 transfected lysate(35.2 KDa).
Lane 2: Non-transfected lysate. Mouse Purified Hu E(detection), WB
50 µg / €370.00
  Abnova Taiwan Corp.

EIF4A3 antibody

Immunoperoxidase of monoclonal antibody to DDX48 on formalin-fixed paraffin-embedded human salivary gland. [antibody concentration 1 ug/ml] Mouse IgG1 2D12 Purified Hu E, P
50 µg / €370.00
  Abnova Taiwan Corp.

EIF4B pSer422 antibody

Figure 1. Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue using eIF4B pSer422 antibody (AP09472PU) (Left) and the same antibody preincubated with blocking peptide (Right). Rabbit Aff - Purified Hu, Ms P, WB
0.1 ml / €320.00
  Acris Antibodies GmbH

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