Overview

Description

  • Nature
    Recombinant
  • Source
    Baculovirus infected Sf9 cells
  • Amino Acid Sequence
    • Species
      Human
    • Tags
      GST tag N-Terminus

Associated products

Specifications

Our Abpromise guarantee covers the use of ab60835 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

  • Applications

    Functional Studies

    SDS-PAGE

  • Purity
    > 90 % SDS-PAGE.
    Purity: >90% as determined by densitometry.
  • Form
    Liquid
  • Additional notes

    ab204880 (RPS6 peptide) can be utilized as a substrate for assessing kinase activity

  • Concentration information loading...

Preparation and Storage

  • Stability and Storage

    Shipped on dry ice. Upon delivery aliquot and store at -80ºC. Avoid freeze / thaw cycles.

    pH: 7.50
    Constituents: 0.0038% EGTA, 0.00174% PMSF, 0.00385% DTT, 0.79% Tris HCl, 0.00292% EDTA, 25% Glycerol, 0.87% Sodium chloride

    This product is an active protein and may elicit a biological response in vivo, handle with caution.

General Info

  • Alternative names
    • Oncogene PIM 1
    • Oncogene PIM1
    • PIM
    • PIM 1
    • Pim 1 kinase
    • pim 1 kinase 44 kDa isoform
    • Pim 1 oncogene
    • pim 1 oncogene (proviral integration site 1)
    • PIM1
    • pim1 kinase 44 kDa isoform
    • PIM1_HUMAN
    • Pim2
    • PIM3
    • Proto oncogene serine/threonine protein kinase Pim 1
    • Proto-oncogene serine/threonine-protein kinase Pim-1
    • Proviral integration site 1
    • Proviral integration site 2
    see all
  • Function
    May affect the structure or silencing of chromatin by phosphorylating HP1 gamma/CBX3. Isoform 2 promotes the G1/S transition of the cell cycle via up-regulation of CDK2 activity and phosphorylation of CDKN1B, resulting in enhanced nuclear export and proteasome-dependent degradation of CDKN1B. Isoform 2 also represses CDKN1B transcription by phosphorylating and inactivating the transcription factor FOXO3. Plays a role in signal transduction in blood cells. Contributes to both cell proliferation and survival and thus provides a selective advantage in tumorigenesis.
  • Tissue specificity
    Expressed primarily in cells of the hematopoietic and germline lineages. Isoform 1 and isoform 2 are both expressed in prostate cancer cell lines.
  • Sequence similarities
    Belongs to the protein kinase superfamily. CAMK Ser/Thr protein kinase family. PIM subfamily.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications
    Autophosphorylated on both serine/threonine and tyrosine residues.
  • Cellular localization
    Cytoplasm. Nucleus and Cell membrane.
  • Information by UniProt

Images

  • Sample Kinase Activity Plot.
  • ab60835 on SDS-PAGE, MW ~62kDa.

References

ab60835 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question
Answer

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.

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Question
Answer

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.

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Answer

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.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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