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Dear technical support team: This customer has purchased ab70 (Anti-HPV16 E6 + HPV18 E6 antibody [C1P5]) and has conducted the WB several times. The results show no signal in HPV16 E6; therefore this customer wants to ask for your help to modify her experiment step, could you please offer any suggestion to improve her wb result? I attached the wb image in this letter and her experiment step as follow: 1. Order details: Batch number: gr45058-1 Po:943411 Abcam product code: ab70 Antibody storage conditions (temperature/reconstitution etc) -20 2. Please describe the problem (high background, wrong band size, more bands, no band etc). I used the anti-16E6/18E6 antibody (Ab70) to check the recombinant protein of 16E6 or 18E6 at the same time. It seems it can detect the 18E6 protein but not the 16E6 protein. 3. On what material are you testing the antibody in WB? · Species: E.coli ( rosetta, constructed into pGEX4T-1 vector) or Caski cell lysate (Human) · What’s cell line or tissue Caski cell · Cell extract or Nuclear extract: cell extraction · Purified protein or Recombinant protein: Recombinant protein or cell lysate 3. The lysate How much protein was loaded: at least 35 ug/well or the E.coli pellet after IPTG induction (with SDS sample buffer to denature What lysis buffer was used: Pierce E. coli extraction buffer or cell lysis reagent What protease inhibitors were used: PIC(Protease Inhibitor Cocktail from Pierce) What loading buffer was used: 6 X SDS sample buffer Phosphatase inhibitors: No Did you heat the samples: temperature and time: 100℃ for 5-10 min 4. Electrophoresis/Gel conditions/ Transfer conditions Reducing or non reducing gel: SDS-PAGE, denature form Reducing agent:6 X SDS-PAGE sample buffer Gel percentage : 12.5% Transfer conditions: (Type of membrane, Protein transfer verified): PVDF, 400 mA transfer 2hr 5. Blocking conditions Buffer:PBST Blocking agent: milk, BSA, serum, what percentage: 5% silk milk in PBST Incubation time:4℃ overnight Incubation temperature: 4℃ 6. Primary Antibody Species:mouse Reacts against: anti-16E6/18E6 (Human papillomavirus E6 protein) · At what dilution(s) have you tested this antibody:1:1000 · What dilution buffer was used: PBS · Incubation time: 1hr · Incubation temperature: RT · What washing steps were done: PBST wash 3 times at RT 7. Secondary Antibody Species:sheep Reacts against:anti-mouse IgG At what dilution(s) have you tested this antibody: 1:1000 Incubation time: 1hr Wash steps: PBST wash 3 times at RT Fluorochrome or enzyme conjugate: peroxidase Do you know whether the problems you are experiencing come from the secondary? No 8. Detection method ECl, ECl+, other detection method:ECL 9. Did you apply positive and negative controls along with the samples? Please specify. Ans: I used the anti-16E6/18E6 antibody (Ab70) to check the recombinant protein of 16E6 or 18E6 at the same time(18E6 as positive control, pre-induction pellet as negation control). It seems can detect the 18E6 protein but can't detect the 16E6 protein. I have already confirmed the sequence of 16E6 construct is correct. 10. Optimization attempts · How many times have you tried the Western? At least 3 times · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): No · Do you obtain the same results every time e.g. are background bands always in the same place? yes · What steps have you altered? extend the incubation time to overnight at 4℃ Thanks for your kindly help. Best regards
Asked on Oct 28 2011
Thank you for your enquiry regarding ab70 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody. Additionally, thank you for supplying an image, as this has been very beneficial in understanding your concerns. The protocol looks fine to me however I would like to make few comments/suggestions that might help to improve your results and that you could consider trying: As the antibody gave expected results with 18E6 protein so I can say the antibody is fine. However I would suggest optimizing the protocol for 16E6 protein. Could you try either high concentration of 16E6 lysates or low dilution e.g. 1/500 of ab70? If the results do not improve please do not hesitate to contact me; I will then send you a free of charge replacement vial of same antibody.
回复于 Oct 28 2011