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Dear technical support team: This customer has purchased ab11048 (Anti-CDKN2A/p14ARF antibody [ARF 4C6/4]), and he raised an enquiry about his ICC data which was staining in cell nucleus. However, according to the online reference indicated this protein should expression in cell nucleolar. Therefore, this customer would like to ask for your help to offer some suggestion to him. I also attached his ICC data in this letter, and his experiment step as follow: 1) Order details: ˙Batch number ( Lot number): GR37995-1 ˙Abcam product code: ab11048 ˙Antibody storage conditions (temperature/reconstitution etc) :-20℃ 2) Please describe the problem (high background, non-specific signal…etc). The signal should in nucleolar 3) On what material are you testing the antibody in IHC/ICC? ˙Species: human ˙What’s cell line or tissue: U2OS cell 4) Sample preparation: ˙Type of sample (Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed ˙paraffin embedded sections, cells in culture, other:____) : cell culture ˙Sample preparation: cells seed in coverslip 5) Fixation step ˙Yes or No : yes ˙If yes, Fixative agent and concentration: 4% paraformaldehyde ˙Fixation time: 10min ˙Fixation temperature : room templature 6) Antigen retrieval method(including time, temperature etc.): no 7) Permeabilization method: ˙Permeabilizing agent and concentration: 0.5% Triton X-100 in PBS 8) Blocking agent (eg BSA, serum…): no blocking 9) ˙Endogenous peroxidases blocked? no ˙Endogenous biotins blocked? no 10) Primary antibody (If more than one was used, describe in “additional notes”) : ˙Species: mouse monoclonal ˙Reacts against: p14ARF ˙At what dilution(s) have you tested this antibody: 1:25 ˙Diluent buffer: PBS ˙Incubation time: overnight ˙Incubation temperature: 4℃ ˙What washing steps were done (which buffer, number of washes): PBS wash three times 11) Secondary antibody: ˙Species:goat ˙Reacts against which species: mouse ˙At what dilution(s) have you tested this antibody: 1:100 ˙Diluent buffer:PBS ˙Incubation time:1 hour in RT ˙What washing steps were done (which buffer, number of washes): PBS wash three times ˙Fluorochrome or enzyme conjugate (eg: FITC, HRP, AP, biotin…etc):FITC ˙Do you know whether the problems you are experiencing come from the secondary? The secondary antibody is working in other experiment 12) Signal amplification method (eg: ABC, LSAB, HRP polymer, TSE): no 13) Detection method (eg: DAB, BCIP/NBT …etc): no 14) ˙ How many times have you run this staining? Three times ˙Do you obtain the same results every time? Yes ˙What steps have you altered to try and optimize the use of this antibody? 1. Fixation buffer(paraformaldehyde, formaldehyde or acetone) 2. incubation condition of first antibody Could you please help this customer to solve the problem? Thanks for your kindly help Best regards
Asked on Oct 12 2011
Thank you for contacting Abcam Technical Team 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 product. According to Swiss-Prot, the localization of the staining (CDKN2A/p14ARF) could be both cytoplasmic and nuclear. You may wish to take a look at this site for further information: http://www.uniprot.org/uniprot/P42771 U2OS is a human osteosarcoma cell line; it is known that CDKN2A/p14ARF is widely expressed but not detected in certain tissue such brain or skeletal muscle. There are at least 4 different splice variants and the expression pattern of these isoforms (P42771-1, P42771-2, P42771-3, Q8N726-1) may vary in different tissue/cell types. After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions: 1) Try to use cells which are intensively dividing and the cell culture should in exponential (log) phase of growth. 2) It may be worth staining HeLa cells or sections of human liver or lymphomas as good positive control. 3) In order to increase the signal, it would be worth applying ABC signal enhancing system. I hope this helps and if I can assist further, please do not hesitate to contact me.
Answered on Oct 12 2011