Anti-PRDM1/Blimp1 antibody (ab13700)

Thank you for your enquiry. We do not have any data that the antibody shows the 2 isoforms on western blots of myeloma cell lysates. We only have the data on the lymphoma specimen. As long as the amino acids 778-789 (KVKQETVEPMDP) are present in both isoforms and the proteins can be separated sufficiently on an SDS-PAGE gel, then I do not see why this antibody should not be able to detect both isoforms. Of course, as I mentioned, we have not formally tested this. I hope this information helps, please do not hesitate to contact us if you need any more advice or information,

I have received a nice reply from the supplier of the ICC image. He states "We simply used a very standard protocol. Fixed for 20 mins at 4C in 4%PFA, blocked in 10% donkey serum for about 60 mins at room temp, then added primary overnight at 4C, then secondary for 4 hours at 4C". I hope this information helps. Please do not hesitate to contact me should you require further assistance.

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with ab13700. The ICC image on the datasheet was submitted by a customer who wishes to remain anonymous. I have contacted him directly requesting further information if there was anything out of the ordinary done to get this antibody working in ICC. I will contact you in due course should any details emerge. With regards to a positive control, following some literature searching I came across: Chang DH, Angelin-Duclos C, Calame K. BLIMP-1: trigger for differentiation of myeloid lineage. Nat Immunol. 2000 Aug;1(2):169-76. In the paper they treated promyeloid leukemia cell lines U937 and HL60. By inducting them to differentiate using PMA they directly upregulated BLIMP1, detectable using ICC and the ribonuclease protection assay. Therefore I think that if you could get hold of one of these cell lines and perhaps monitor their BLIMP1 mRNA levels to confirm upregulation they would serve as excellent positive controls. Should this not be of assistance I would encourage you to submit a technical questionnaire by clicking on the link below. This will better give us an appreciation of the optimisation steps that you have undertaken. https://www.abcam.com/index.html?section=ihc&pageconfig=technical&intAbID=13700&mode=questionaire

Thank you for your reply and for sending us further information about your customer's Western blot protocol. Looking through the original completed Questionnaire and the forwarded extra-information you have just sent to us, I would like to make the following suggestions: As the datasheet of this antibody indicates, ab13700 has been tested and characterised in human lymph node lysate. 35µg total protein per lane of this type of lysate was used (prepared in RIPA buffer) and the antibody at a concentration of 1µg/ml was incubated with the membrane. Unfortunately, we do not have information about the cell line (RPMI 8226) your customer is using. Therefore we would strongly suggest using human lymph node lysate as a well characterised positive control. For Western blot application, it is extremely important to determine the protein concentration of the samples. The customer should load 20-30 total protein per lane. The lysis buffer is also important, and we would recommend your customer using RIPA buffer: Preparation of RIPA cell lysates for Western Blots: 1. Wash cell pellets once with ice-cold PBS. 2. Add 1 ml of RIPA buffer to 10x8 cells, incubate on ice for 20 min, vortex 2 to 3 times. 3. Centrifuge for 5 min at 4oC at maximum speed in a microfuge tube. 4. Transfer supernatant into clean tube. Measure protein concentration with a protein assay. 5. Adjust concentration to 5 mg/ml with RIPA lysis buffer. 6. Add equal volume of 2 x SDS sample buffer into cell lysate, boil for 5 min. 7. Store at -20oC for daily use or -80oC for long term. Avoid repeated freeze thaw cycles. RIPA Base Ingredients Tris-HCl: 50 mM, pH 7.4 NP-40: 1% Na-deoxycholate: 0.25% NaCl: 150 mM EDTA: 1 mM PMSF: 1 mM Aprotinin, leupeptin, pepstatin: 1 microgram/ml each RIPA Protease Inhibitors Phenylmethylsulfonyl fluoride (PMSF) (200 mM stock solution in isopropanol; store at room temperature) EDTA (calcium chelator; 100 mM stock solution in H2O, pH 7.4) Leupeptin (store frozen in aliquots, 1 mg/ml in H2O) Aprotinin (store frozen in aliquots, 1 mg/ml in H2O) Pepstatin (store frozen in aliquots, 1 mg/ml in methanol) We hope this information will be useful.