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Hi, i'm responding the questionary 1) Abcam product code ab: ab13413, ab27710, ab77811 2) Abcam order reference number or product batch number 3) Description of the problem: no bands 4) Sample preparation: Type of sample: HepG2 whole cell lysates Species : Human Lysis buffer : 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol Protease inhibitors: aprotinin, leupeptin, bestatin, pesptatin and E-64 Phosphatase inhibitors : Reducing agent : DTT Boiling for ≥5 min? yes Protein loaded ug/lane: generally 8 ug Positive control : Negative control : 5) Percentage of gel: 12% Type of membrane: Nitrocelulose Protein transfer verified: Panceau Red Blocking agent and concentration: I get no bands with non fat milk 5% + TBST, and some ghosty bands with BSA sigma 5% + TBST Blocking time: 2h for milk and overnight for BSA Blocking temperature: 4 oC 6) Primary antibody (If more than one was used, describe in “additional notes”) : Concentration or dilution generally 1:1000 Diluent buffer TBST + blocking agent Incubation time O.N. utilizing milk 5% as blocking agent and 2h utilizing BSA 5% as blocking agent. Incubation temperature: 4 oC 7) Secondary antibody: anti-mouse Species: Reacts against: Concentration or dilution , generally 1:4000 Diluent buffer TBST + blocking agent Incubation time 2h Incubation temperature: 4 oC Fluorochrome or enzyme conjugate: HRP 8) Washing after primary and secondary antibodies: Buffer TBST Number of washes 3x (10 min) 9)Detection method ECL 10) How many times have you run this staining? 3 Do you obtain the same results every time? yes What steps have you altered to try and optimize the use of this antibody? Change the blocking agent (Milk to BSA).
Asked on Dec 02 2011
Thank you for getting back to me with those details. Having reviewed your protocol there are a few things I may be able to suggest. The dilution for ab13413 is recommended in the range of 5-10µg/ml and ab27710 at an assay dependent dilution. I would therefore suggest decreasing the dilution that you are using trying 1/200 and 1/500 to see if any improvement is observed. Even though ab77811 is recommended at 1/1000 I would also suggest trying this with 1/200 and 1/500 as this may also need optimising with your sample. I would also suggest increasing the amount of protein that you are loading on the gel, we would normally recommend loading between 20-40 µg per well. As you are observing some bands with blocking using BSA I would continue to use this blocking agent. Initially using 3% in TBST incubating for 1-2 hours at room temperature. I would then incubate the primary antibody diluted in 1% BSA TBST for 1 hour at room temperature and overnight at 4°C with agitation. It may be that the conditions that you have been using has been over blocking the membrane. I would also reduce the wash step to 3x5 min. I realise this is alot to try with three different antibodies so I would recommend optimising each one at a time to find the best experimental conditions for each antibody. May I ask what secondary antibodies you have been using to detect the primary antibodies. ab13413, ab2771 and ab77811 are mouse IgG1, IgG2b and IgM respectively and would need to be used with secondary antibodies which are able to recognise these isotypes. I hope these suggestions help to improve the results you have been observing. If you continue to have problems please do let me know.
Answered on Dec 02 2011