Overview

  • Product name
    Anti-EIF2S1 (phospho S51) antibody
    See all EIF2S1 primary antibodies
  • Description
    Rabbit polyclonal to EIF2S1 (phospho S51)
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, WB, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Human, Saccharomyces cerevisiae, Mink
  • Immunogen

    Synthetic peptide (Human) derived from the region of human eIF-2 alpha that contains serine 51. This region is conserved among many species including rat, pig, cow, fruit fly, and yeast.

  • Positive control
    • 3T3-L1 adipocytes stimulated with Leukemia Inhibitory Factor (LIF).

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer
    pH: 7.30
    Preservative: 0.05% Sodium azide
    Constituents: PBS, 50% Glycerol, 0.1% BSA

    BSA is IgG and protease free
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    Purified from rabbit serum by sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated eIF-2 alpha. The final product is generated by affinity chromatography using an eIF-2 alpha-derived peptide that is phosphorylated at serine 52.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab4837 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF Use at an assay dependent concentration.
WB 1/1000. Detects a band of approximately 40 kDa (predicted molecular weight: 36 kDa).
IHC-Fr 1/250. See Abreview.

Target

  • Function
    Functions in the early steps of protein synthesis by forming a ternary complex with GTP and initiator tRNA. This complex binds to a 40S ribosomal subunit, followed by mRNA binding to form a 43S preinitiation complex. Junction of the 60S ribosomal subunit to form the 80S initiation complex is preceded by hydrolysis of the GTP bound to eIF-2 and release of an eIF-2-GDP binary complex. In order for eIF-2 to recycle and catalyze another round of initiation, the GDP bound to eIF-2 must exchange with GTP by way of a reaction catalyzed by eIF-2B.
  • Sequence similarities
    Belongs to the eIF-2-alpha family.
    Contains 1 S1 motif domain.
  • Post-translational
    modifications
    Substrate for at least 4 kinases: EIF2AK1/HRI, EIF2AK2/PKR, EIF2AK3/PERK and EIF2AK4/GCN2. Phosphorylation stabilizes the eIF-2/GDP/eIF-2B complex and prevents GDP/GTP exchange reaction, thus impairing the recycling of eIF-2 between successive rounds of initiation and leading to global inhibition of translation (PubMed:15207627, PubMed:18032499). Phosphorylated; phosphorylation on Ser-52 by the EIF2AK4/GCN2 protein kinase occurs in response to amino acid starvation and UV irradiation.
  • Cellular localization
    Cytoplasmic granule. The cytoplasmic granules are stress granules which are a dense aggregation in the cytosol composed of proteins and RNAs that appear when the cell is under stress. Colocalizes with NANOS3 in the stress granules (By similarity).
  • Information by UniProt
  • Database links
  • Alternative names
    • EIF 2 alpha antibody
    • EIF 2 antibody
    • EIF 2A antibody
    • EIF 2alpha antibody
    • eIF-2-alpha antibody
    • eIF-2A antibody
    • EIF-2alpha antibody
    • EIF2 alpha antibody
    • EIF2 antibody
    • EIF2A antibody
    • EIF2S1 antibody
    • Eukaryotic translation initiation factor 2 subunit 1 alpha 35kDa antibody
    • Eukaryotic translation initiation factor 2 subunit 1 alpha antibody
    • Eukaryotic translation initiation factor 2 subunit 1 antibody
    • Eukaryotic translation initiation factor 2 subunit alpha antibody
    • IF2A_HUMAN antibody
    see all

Images

  • Peptide Competition:

    Extracts prepared from 3T3-L1 adipocytes were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF.

    Membranes were blocked with a 5% BSA-TBST buffer overnight at 4oC, then were incubated with 0.50 µg/mL ab4837 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with:
    1) the phosphopeptide immunogen
    2) the non-phosphopeptide corresponding to the immunogen
    3) no peptide

    After washing, membranes were incubated with goat F(ab)2 anti-rabbit IgG alkaline phosphatase and bands were detected using the Tropix WesternStar method. The data show that only the peptide corresponding to ab4837 blocks the antibody signal, thereby demonstrating the specificity of the antibody.


     

    Peptide Competition:

    Extracts prepared from 3T3-L1 adipocytes were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF.

    Memb

  • ab4837 staining eIF2S1(phospho S51) in mouse skeletal muscle tissue sections (5 µm) by IHC-Fr (Frozen sections). Tissue samples were fixed with acetone (for 10 minutes at -20°C) and blocked with 2% BSA for 1 hour at 25°C. The sample was incubated with primary antibody (1/250 in PBS with 1% BSA) at 4°C for 9 hours. An Alexa Fluor®488-conjugated Goat polyclonal to rabbit IgG (1/500) was used as secondary antibody.

    See Abreview

  • ab4837 staining eIF2S1 in human 293ft cells by Immunocytochemistry/ Immunofluorescence. The cells were paraformaldehyde fixed, permeabilised in 0.1% Triton X-100 and then blocked using 5% serum for 1 hour at 25°C. Samples were then incubated with primary antibody at 1/200 for 2 hours at 25°C. The secondary antibody used was a goat anti-rabbit IgG conjugated to Alexa Fluor® 488 (green) used at a 1/250 dilution. DAPI was used to stain the cell nuclei (blue).

    See Abreview

References

This product has been referenced in:
  • Hans F  et al. Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43). J Biol Chem 293:16083-16099 (2018). Read more (PubMed: 30120199) »
  • Mohler K  et al. Editing of misaminoacylated tRNA controls the sensitivity of amino acid stress responses in Saccharomyces cerevisiae. Nucleic Acids Res 45:3985-3996 (2017). WB . Read more (PubMed: 28168297) »
See all 11 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

Application
Western blot
Sample
Pig Cell lysate - whole cell (endothelial cell)
Gel Running Conditions
Non-reduced Denaturing (8% gel)
Loading amount
30 µg
Specification
endothelial cell
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C

Abcam user community

Verified customer

Submitted Jul 24 2015

Question

Dear Tech support Attached is a customer complaint form. Please advise. Thank you in advance for your kind help. Regards, Details Antibody code: Rabbit polyclonal to EIF2S1 ab4837 Batch number: GR46360 Antibody storage conditions (temperature/reconstitution etc) aliquoted and stored at -20C Description of the problem (high background, wrong band size, more bands, no band etc.) detects only human EIF2S1 and not mouse (as supposed to) and gives one none specific band at about 70kDa. Figure is attached below. Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.) Human – HK-2 (human kidney) cell extract Mouse - kidney extracts Sample preparation (Buffer/Protease inhibitors/Heating sample etc.) Lysates of both kidney tissues and cell extracts were preformed in the same manner: lysis of tissue/ cells in 1% triton with protease inhibitors. After protein concentration was determined, samples were added with sample buffer (with beta-mercaptoethanol) and pre heated 5min at 95C prior to loading. Amount of protein loaded Human cells – 20ug Mouse kidney tissue – 30ug Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.) Reducing gel, 12% lower gel, 4% upper gel Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) Transfer buffer: Tris, glycine, methanol, 1.5hr Blocking agent: 5% BSA in TBST, O.N in 4C Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Ab4837, diluted 1:1000 in 3% BSA solution in TBST, incubation time : 2hr at R.T Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Wash step: 4 washes in TBST, each of 4min Peroxidase-conjuated affinipure goat anti-Rabbit IgG (H+L), diluted 1:50,000 in TBST, 1hr at R.T Detection method (ECL, ECLPlus etc.) ECL Positive and negative controls used (please specify) Both human and mouse samples were loaded on the same gel, as described above. Bands only appeared for human samples and not for mouse samples, implying the entire western procedure was preformed properly, but the antibody did not recognize the mouse samples ( but it is supposed to..). these mouse kidney lysates were tested in other westerns, with different antibodies, and worked well. Optimization attempts (problem solving) How many times have you tried the Western? Only once. Each trial is 10ul out of 50ul given.. Have you run a "No Primary" control? No Do you obtain the same results every time? e.g. are the background bands always in the same place? What steps have you altered? Additional Notes

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Answer

Thank you for your enquiry regarding ab4837 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this antibody. After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions: It is clear that the antibody works fine in human samples and at the dilution of 1/1000 detects human EIF2S1. 1) I can confirm that ab4837 also recognizes mouse EIF2S1 sequence. However, it is important to optimize the working/final concentration of the primary and the expression levels of EIF2S1 in the human and mouse samples could be different. 2) It would also be essential to demonstrate that the lysate has enough membrane material containing EIF2α in both speacies. It maybe that the lysate has enough GAPDH or actin (depends on the internal control he has) but that is notsufficinet to detect the target EIF2α. I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (Brain lysate)
Loading amount
50 µg
Specification
Brain lysate
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Aug 20 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (293FT cells)
Loading amount
100000 cells
Specification
293FT cells
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Jun 02 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (293FT cell line)
Specification
293FT cell line
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton X-100
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Mar 26 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (skeletal muscle)
Specification
skeletal muscle
Fixative
Acetone
Permeabilization
No
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Mar 05 2010

Answer

Thank you for this information and clarification. I have changed the online datasheet to reflect the fact that the antibody is raised against eIF2 alpha. Thank you again for your assistance.

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Answer

Thank you for your enquiry. Our designation comes directly from Swiss-Prot where the synonyms for the protein are as follows: Eukaryotic translation initiation factor 2 alpha subunit eIF-2-alpha EIF-2alpha EIF-2A I believe the datasheet is correct in identifying the protein that the antibody is raised against. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your enquiry. I contacted the originator of this antibody and was provided with the following information. There is some history in the literature regarding this particular sequence. Basically, the serine residue is at position 51 in the Drosophila sequence and at position 52 in the mammalian sequences. "The antibody directed to eIF-2alpha pS51 was raised to a peptide immunogen that contained approximately 10 amino acid residues. In the sequences below, I show approximately where the sequence lies within the published sequences for this protein from several species. The sequences which I have presented below show that the amino acid sequence surrounding the serine of interest is identical between the species. The position of the serine within the protein is just shifted between Drosophila (position 51) and the two mammalian species (position 52). The phosphorylation site specific antibody preparation directed to eIF-2alpha pS51 will react with the phosphorylated protein from various mammalian species, as well as with the phosphorylated protein from Drosophila, due to the conservation of the sequence. The peptide controls have the exact sequence of the peptide immunogen. The positive control peptide, the one that should compete with the eIF-2alpha for antibody binding sites, is phosphorylated at the serine residue. The negative control peptide, the one which should not compete with the eIF-2alpha for antibody binding sites, lacks the phosphate group on the serine residue of interest. As I was going through our literature on this subject, I noticed that we have a paper that cites the use of this antibody in the detection of phosphorylation of eIF-2alpha in Saccharomyces cerevisiae. I tried to find the sequence in the literature for eIF-2alpha from this species, but it appears that it is not yet published. The observation that our antibody reacts with it by Western blotting suggests that there is sequence conservation between fly, human, rat, and yeast." I hope this helps to explain things. Please let me know if you need additional assistance. AAA53627: Drosophila melanogaster ORIGIN 1 maltsrfyne rypeiedvvm vnvlsiaemg ayvhlleynn iegmillsel srrrirsink 61 lirvgktepv vvirvdkekg yidlskrrvs pedvekcter fakakainsl lrhvadilgf 121 egnekledly qktawhfekk ynnktvaydi fkqsvtdptv fdecnlepet kevllsnikr 181 klvsptvkir adiecscygy egidavkasl tkglelstee lpirinliap plyvmttstt 241 kktdglkale vaiehirakt seydgefkvi mapklvtaid eadlarrler aeaenaqvag 301 dddeedgadq egmqfdpeke fnhkgsgagr aneedeeeee d AAA41110: Rat ORIGIN 1 mpglscrfyq hkfpevedvv mvnvrsiaem gayvslleyn niegmillse lsrrrirsin 61 klirigrnec vvvirvdkek gyidlskrrv speeaikced kftksktvys ilrhvaevle 121 ytkdeqlesl fqrtawvfdd kykrpgygay dafkhavsdp sildsldlne derevlinni 181 nrrltpqavk iradievacy gyegidavke alraglncst etmpikinli appryvmttt 241 tlerteglsv lnqamavike kieekrgvfn vqmepkvvtd tdetelarql erlerenaev 301 dgdddaeeme akaed AAA52373: Human ORIGIN 1 mpglscrfyq hkfpevedvv mvnvrsiaem gayvslleyn niegmillse lsrrrirsin 61 klirigrnec vvvirvdkek gyidlskrrv speeaikced kftksktvys ilrhvaevle 121 ytkdeqlesl fqrtawvfdd kykrpgygay dafkhavsdp sildsldlne derevlinni 181 nrrltpqavk iradievacy gyegidavke alraglncst enmpikinli appryvmttt 241 tlerteglsv lsqamavike kieekrgvfn vqmepkvvtd tdetelarqm erlerenaev 301 dgdddaeeme akaed

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Answer

Thank you for your enquiry. Here are a few thoughts to consider: 1) EIF2a is a protein involved in translation of mRNA messages into protein. As such, I would look for it in the cytoplasm. 2) If someone is looking at serine phosphorylation, I would recommend to make as few manipulations as possible before getting the sample into a buffer that protects the phosphorylation site. Making a nuclear extract may work against this. 3) It may be more useful to look at your blotting conditions and whether there should be any phosphorylation of Eif2a in the sample in the first place. They may be overloading/overexposing blots in order to try to see something that is not there. 4) If you are looking for extremely low levels of serine phosphorylation, you may wish to run an IP eperiment instead of a nuclear extract. 5) remember to block with BSA 5% rather than milk when blotting for phospho-proteins. I hope this helps. Do not hesitate to contact us again if you need further assistance,

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1-10 of 12 Abreviews or Q&A

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