Anti-Separase 抗体 [XJ11-1B12] (ab16170)

Thank you for calling us and for alerting us to the problem you are experiencing with our product. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly. If you wouldn't mind, could you please fill in the questionnaire with details of the experiments where ab16170 was used. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody. In regards to the reactivity of this antibody with chicken, unfortunately having compared the human (SwissProt Q14674) separase to that of chicken (NCBI XP_423289.2) the alignment is very poor and I would therefore not expect this antibody to detect the chicken protein. All the other antibodies we have for this target are also raised against the human protein and I can therefore not recommend an alternative. You may have more luck by searching using biocompare: www.biocompare.com If you have any further questions please do not hesitate to ask.

Thank you for your enquiry. Here we are sending a general Western blot protocol. The information is only intended as a guide. The researchers should determine what protocol best meets their needs: Sample Preparation 1. Remove media and wash cells with sterile PBS. 2. Scrape cells into PBS and pellet in a centrifuge. 3. Resuspend pellet in 300 ul of RIPA buffer. 4. Pass lysate through 21 gauge needle 3-4 time to shear the DNA. 5. Incubate on ice for 30-60 min. 6. Spin at 15,000g for 20 minutes at 4ºC. 7. Remove and keep the supernatant. 8. Measure protein content in supernatant. 9. Add loading buffer to supernatant (50 ml/200 ml supernatant). 10. Add mercaptoethanol (5 ml/100 ml). 11. Boil samples for 5-10 minutes in heating block. II Sample Running and Transfer: 1. Follow recommendations of manufacture. 2. Verify transfer by staining with Ponceau S dye. 3. Mark blot with pencile III Detection: 1. Block blot in 5% milk/TBS/Tween in the container, 1hr, RT, on shaker; OR o/n in 4C on a rocker. 2. Dilute primary antibody in 5% milk/TBS and incubate with blot for 1hr RT, or o/n in cold room on a rocker. 3. Wash: 3 x 5 minutes in TBS/Tween. 4. Dilute secondary antibody in 5% milk/TBS, incubate 1hr at RT, on shaker. 5. Wash: 3 x 10 minutes in TBS/Tween. 6. Cut a pc. of transparency into 2 smaller pcs. that fit into the cassette; mix oxidizing and luminol reagent 1: 1, transfer onto a piece of parafilm, lay the blot over the solution for 1 min; then insert the blot between the 2 transparencies. Make sure to remove all bubbles. 7. Initially expose for 1 min and then adjust for the later trial. The signals will decay over time. The positive controls used for this experiment were 15 ug of non-induced 5C6 and induced 5C6 whole cell lysates. Both lysates gave a clear positive signal. The induced form gave a stronger signal, but the non-induced form was still very clear. We hope that this helps.