Anti-EIF2S1 (phospho S51) antibody (ab4837)

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

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.

No, Swissprot, EBI's and NCBI's websites are riddled with mistakes and faulty information. So, here's the link you should look at:

http://www.chem.qmul.ac.uk/iubmb/misc/trans.html

It is IUBMB that has the naming rights to translation factors, no one else.

Thank you for your time and letting help you correct other's mistakes.

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.

The antibody products stated to be anti eIF-2A really against eIF2alpha. They are two entirely different proteins.

ab4837 ab5369 ab19288

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.

I was just wondering about ab4837, to phospho-eIF2alpha. We have an old aliquot and it says rabbit polyclonal to eIF-2A (phospho S51), whereas the new antibody tube says rabbit polyclonal to eIF2A (phospho S52). Why the difference between Serine residues, S51 compared to S52 on the new aliquot? Will it matter for my experiment?

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

Just a quick question, I know your protocol for sample preparation for ab4837 is a general lysis buffer similar to RIPA, but would a cytoplasmic extraction prep work for this Ab rather than total cell lysate prep, which I find sometimes give quite dirty blots? Regards,

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,