Anti-PIM1 抗体 [ZP003] (ab54503)

Thank you for contacting us. Here are my answers to your questions. Is there a loading limit (cell number) for sample buffer as the lysis buffer? We recommend 1 ml of lysis buffer per 10 million cells. For sample buffer, if you mean gel loading buffer (for example, Laemmli's), that number of cells should be fine too. Which sample preparation method (lysis buffer vs. sample buffer) is better? Using a lysis buffer such as RIPA without reducing agents allows you to check the protien concentration, which may be useful for loading the gel lanes with a consistent amount of protein and for comparing results from multiple gels. For just simply obtaining bands in the blot, either approach is valid. I would like to check the protein level in another cell type. Which one is better for pim1 and CLK expression? For PIM1, we recommend a lysate of K562, such as ab29306: Click here (or use the following: https://www.abcam.com/K562-whole-cell-lysate-ab29306.html). For CLK, it will depend on which CLK you are trying to detect. I searched the precast gel with largest loading volume. The max. loading volume is 45ul for 12 wells gel. There is limit amount of cells one can lysis per ml of lysis buffer. If I increase the cell amount, the volume of lysis buffer will be increased too. This will not help much with the low expression protein. Can I add sample buffer to unlimited cell numbers? Also there is limit on sample loading on the gel. If your samples have low amounts of PIM1, you may need to immunoprecipitate the protein to enrich for it. The optimal range of protein per gel lane will depend on the gel but the typical range for cell lysates is 20 - 100 ug per lane. Would you please instruct me on the sample preparation method for control protein? Cell lysates should generally be prepared in a lysis buffer containing detergent, such as RIPA, and protease inhibitors. Simply preparing the lysates in your gel loading buffer can be effective too, but gives less control over how much protein, by weight, you load per lane.If I understand you correctly, your control protein is giving you the expected signal in your blots, so that your current protocol is effective. Please reply if that is not the case. Can I use ab60835 (control protein) as the sample instead of cell lysate for antibody screening? Antibodies that are capable of detecting ab60835 may not be capable of detecting endogenous protein in your samples of interest, as you have found. But for an initial screen, yes, the control protein will at least identify antibodies that are capable of detecting human PIM1. My sample is very viscous. Some band are smiling on the gel. After thaw the sample, I centrifuged it. But there is no pellet when I transfer the solution to the new tube. Sonication is the only way to try? Can you please tell me how you prepare your samples before freezing? What does the buffer contain? Sonication of cell lysates on ice will help with viscous DNA. I suggest comparing two samples, +/- sonication. The smiling may be a consequence of using too much voltage when you run your gel, so you may want to try reducing that too.

Thank you for contacting us with your question. It will not make a difference if the host of your secondary antibodies is the same or different, however if you will be using two primaries from different species on the same tissue, it will be best to use pre-adsorbed secondary antibodies. Secondary antibodies that have been pre-adsorbed are treated to remove any cross-reactive molecules, so that the anti-rabbit secondary will not unintentionally react with the mouse primary antibody, and vice versa. I would be happy to help you select some secondary antibodies; what kind of flourophores would you like to use? I look forward to hearing from you. Please let me know if you have any questions.