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We are trying both the immunohistochemistry and the western. While the immuno is promising and hopefully we will be able to come back with something soon; we are having some issues with the western. We tried the dilution of 1:1000 ING4 and 1:5000 secondary and did not get any bands. Can you give us the standardised method that your lab had used to confirm the use as Western including what secondary atibody is recommended along with the dilution. We did the loading control (tubulin) and the loading control looks allright. Will also be grateful if you can attach the western blot pictures for our ready reference.
Asked on Dec 21 2005
Thank you for your enquiry. I'm glad to hear that the antibody is giving good preliminary results in IHC. I hope that you will submit an Abreview with your results. Regarding the Western blot, the recommended starting dilution range is between 1/500 to 1/3000. It may be that you must use the antibody at a higher concentration (I generally recommend starting at 1/500 for polyclonals as well) to visualize the bands. It would be great if you could let me know further details of your protocol so we could see what kind of samples, how much protein, etc. The questionnaire for this is at the following link: https://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=3714&mode=questionaire This antibody was used at 1.0 µg/ml to detect ING4 present in a U2OS whole cell lysate over expressing the protein. A single band was detected at ~29 kDa corresponding to ING4. A 4-20% TRISglycine gradient gel was used to separate the protein by SDS-PAGE under reducing conditions. The protein was transferred to nitrocellulose using standard methods. After blocking using 5% non-fat dry milk in PBS, the membrane was probed with the primary antibody overnight at 4° C followed by washes and reaction with a 1:20,000 dilution of IRDye800 conjugated Rabbit-anti-Goat IgG [H&L] for 45 min at room temperature. LICOR's Odyssey® Infrared Imaging System was used to scan and process the image. Other detection systems will yield similar results. The HRP-conjugated secondary recommended for this antibody is ab6741, Rabbit polyclonal to Goat IgG. I have added a new Western blot image to the datasheet (it may take a couple of hours for it to show up online). Unfortunately I am unable to attach the images to this email, but I have provided a link directly to the datasheet at the bottom of this email. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.
Answered on Dec 22 2005