Recombinant human PIM1 protein (ab60835)

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/index.html?datasheet=29306). 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 your call yestserday and for your patience while I have looked into your enquiry. This protein has not been pre-boiled, so to run a standard reduced, denatured Western blot, you can add the PIM1 protein to loading buffer such as Laemmli and boil as you normally would. When using purified proteins in Western blot, we recommend loading between 10-100 ng of protein per lane. Since ab60835 contains a large N-terminal tag, the protein runs around 62 kDa compared to the 35 and 45 kDa endogenous isoforms. I hope this information will be useful, but please let me know if you have any further questions and I'll be happy to help.

The Pim1 antibodies we have in our catalog are : ab54457 : the epitope recognized by this antibody maps to a region between aa 250-313 and is specific to Pim-1. No cross-reactivity with Pim-2 or -3 is expected. ab74047 and ab74118 can't react with human Pim2, but the homology is 100% with human Pim3. ab75776 : This antibody is not predicted to cross react with Pim2 or Pim3. Sequence analysis shows that the immunogen shares 45% and 53% identity with Pim2 and Pim3, respectively. However, it is not guaranteed without data. ab85898 : this antibody can't detect Pim2 and Pim3, it is specific to Pim1. ab117518 : the epitope recognized by this monoclonal antibody has not been mapped so I cannot provide you with blast homology scores. The Pim1 protein we have in our catalog and which can be used as a positive control is : ab60835 The prices for each antibody as well as for the protein are indicated on the product datasheet (make sure you select the right country using the drop menu at the top right corner of the Abcam website). I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.