Overview

  • Product name
  • Description
    Rabbit polyclonal to DRAK2
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide:

    CSKRFRFDDSLPNPHE

    , corresponding to amino acids 351/365 of Human DRAK2. (Peptide available as ab8454.)

  • Positive control
    • Wholecell lysate from Jurkat cells An approximately 45 kDa band can be detected.
  • General notes


    Apoptosis is mediated by death domain containing adapter molecules and a caspase family of proteases. Certain serine/threonine protein kinases, such as ASK-1 and RIP,are mediators of apoptosis. Two novel serine/threonine kinases that induce apoptosis were recently identified and designated DRAK1 and DRAK2 (for DAP kinase-related apoptosis-inducing protein kinases) (1). DRAKs contain anN-terminal kinase domain and a C-terminal regulation domain. Overexpression of DRAK2 induces apoptosis. DRAKs have high sequence homology to DAP and ZIP kinases, and they represent a novel family of serine/threonine kinases, which mediates apoptosis through their catalytic activities. DRAK2 is located in nucleus and the messenger RNA was ubiquitously expressed in human tissues.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C.
  • Storage buffer
    Preservative: 0.02% Sodium azide
  • Concentration information loading...
  • Purity
    Ion Exchange Chromatography
  • Purification notes
    DRAK2 Antibody is Ion exchange chromatography purified.
  • Primary antibody notes
    Apoptosis is mediated by death domain containing adapter molecules and a caspase family of proteases. Certain serine/threonine protein kinases, such as ASK-1 and RIP,are mediators of apoptosis. Two novel serine/threonine kinases that induce apoptosis were recently identified and designated DRAK1 and DRAK2 (for DAP kinase-related apoptosis-inducing protein kinases) (1). DRAKs contain anN-terminal kinase domain and a C-terminal regulation domain. Overexpression of DRAK2 induces apoptosis. DRAKs have high sequence homology to DAP and ZIP kinases, and they represent a novel family of serine/threonine kinases, which mediates apoptosis through their catalytic activities. DRAK2 is located in nucleus and the messenger RNA was ubiquitously expressed in human tissues.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Light chain type
    unknown
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab8419 in the following tested applications.

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

Application Abreviews Notes
WB 1/500 - 1/1000. Detects a band of approximately 45 kDa (predicted molecular weight: 43 kDa).Can be blocked with Human DRAK2 peptide (ab8454).
ICC/IF Use a concentration of 10 µg/ml.

Target

  • Function
    Acts as a positive regulator of apoptosis.
  • Tissue specificity
    Highly expressed in placenta, lung, pancreas. Lower levels in heart, brain, liver, skeletal muscle and kidney.
  • Sequence similarities
    Belongs to the protein kinase superfamily. CAMK Ser/Thr protein kinase family. DAP kinase subfamily.
    Contains 1 protein kinase domain.
  • Post-translational
    modifications
    Autophosphorylated.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • DAP kinase related apoptosis inducing protein antibody
    • DAP kinase related apoptosis inducing protein kinase 2 antibody
    • DAP kinase-related apoptosis-inducing protein kinase 2 antibody
    • Death associated protein kinase related 2 antibody
    • DRAK 2 antibody
    • DRAK2 antibody
    • Serine/threonine kinase 17b antibody
    • Serine/threonine kinase 17b apoptosis inducing antibody
    • Serine/threonine protein kinase 17B antibody
    • Serine/threonine-protein kinase 17B antibody
    • ST17B_HUMAN antibody
    • STK 17B antibody
    • Stk17b antibody
    see all

Images

  • Lane 1 : Anti-DRAK2 antibody (ab8419) at 1 µg/ml (DRAK2 antibody)
    Lane 2 : Anti-DRAK2 antibody (ab8419) at 2 µg/ml (DRAK2 antibody)

    All lanes : Raji cell lysate

    Predicted band size: 43 kDa

  • All lanes : Anti-DRAK2 antibody (ab8419) at 1/500 dilution

    Lane 1 : Jurkat whole cell lysate with absence of blocking peptide
    Lane 2 : Raji whole cell lysate with absence of blocking peptide
    Lane 3 : Jurkat whole cell lysate with presence of blocking peptide
    Lane 4 : Raji whole cell lysate with presence of blocking peptide

    Predicted band size: 43 kDa
    Observed band size: 45 kDa
    why is the actual band size different from the predicted?



    We are unsure about the nature of the 70 kDa band. However, DRAK2 is autophosphorylated and it is possible that this band corresponds to the phosphorylated form. The fact that the detection of this band is blocked by DRAK2 peptide indicates that it probably is closely related to the DRAK2 protein.
  • ab8419 at 10µg/ml staining DRAK2 in Jurkat cells by ICC/IF

References

ab8419 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Abreviews
Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (B16F10 murine melanoma cell line)
Loading amount
40 µg
Specification
B16F10 murine melanoma cell line
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 4%

Abcam user community

Verified customer

Submitted Aug 04 2006

Answer

Cross-reaction for peptide antibodies is not uncommon, the band at 42 kDa is specific to DRAK2, but the 70 kDa band is likely to be against a different protein from our own internal testing.

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Answer

Thank you for your enquiry. It is unnecessary to isolate nuclear proteins before running a western blot. You can detect DRAK2 by loading the Jurkat whole cell lysate directly onto the gel.

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Question

Message 1. I have a problem to detect the 45 kDa band of DRAK2 in western blot. Positive control shows only a 70 kDa band which shell not be related to DRAK2. What can I do? You support Jurkat lysate for positive control. DRAK2 is a protein of nucleus. Which procedure do you prefer for blotting and blocking? Message 2. I filled the answers to your questions into the text. I can not send a image, sorry. Thanks for your quickly answer. 1. Please describe the problem (high background, wrong band size, morebands, no band etc). There is only one band at 70 kDa and no 45 kDa band is seen 2. On what material are you testing the antibody in WB? • Species? • Cell extract/ Nuclear extract? • Purified protein? • Recombinant protein? I was testing the antibody on human nuclear extracts and Jurkat cell lysate ab 7889. 3. How much protein did you load? 30 ug nuclear protein • How did you prepare the lysate for the analysis (protease inhibitors etc)? • Did you heat the samples? I heat the samples with Laemmli buffer for 5 min in a boiling wather bath 4. Primary Antibody • Specification (in which species was it raised against)? • At what dilution(s) have you tested this antibody? • Incubation time, wash step? Your anti-DRAK2 antibody was raised against human DRAK2. It was inkubated over night a 4 °C in a 1:500 dilution. After incubation membrane was washed 3 times for each 10 min in TBS containing 0.5%Tween 20 and 1% non fat dry milk. 5. Secondary Antibody • Specification (in which species was it raised against)? Goat anti rabbit IgG antibody • At what dilution(s) have you tested this antibody? • Incubation time, wash step? • Do you know whether the problems you are experiencing come from the secondary? Secondary antibody was raised against IgG, incubated for 1 hour at room temperature. Wash steps like primary antibody 6. What detection method are you using? I used HRP-detection. 7. Background bands • Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control) • Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4°C with agitation) • Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) • At what size are the bands migrating? Could they be degradation products of your target? • Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) There are no bachground bands. So I'm sure that blocking and washing steps are sufficient. Only a strong band at 70 kDa was seen in Jurkat cell lysate and samples. 8. Optimization attempts • How many times have you tried the Western? • Do you obtain the same results every time e.g. are background bands always in the same place? • What steps have you altered? I have it tried for to times. First time there were bachground bands. Second time I modified the wash step. If I washed membrane with TBS, Tween and 1% dry milk and incubated antibodys in TBS, Tween and 5 % dry milk the unspecific bands were lost and only this 70 kDa band was detected. 9. Did you apply positive and negative controls along with the samples? Please specify. Positive control was the Jurkat cell lysate (see above). I do not apply a negative controll.

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Answer

Thank you for your enquiry. The larger band is probably a cross-reaction to a non-related protein or a different member of the same family. We would suggest trying longer exposure of the membrane to x-ray film (30 minutes to overnight). It is also possible that the detection enzyme may be inactivated. Sodium azide inactivates horseradish peroxidase (HRP) irreversibly. Also, bacterial contamination also diminishes HRP activity. Polyvinyl wrap from some sources has been found to hide the signal. You could repeat the incubation with chemiluminescence reagents and place the blot between two pieces of acetate transparency film. Western Blot Protocol: 1) Load 20 to 25 microgram of whole cell lysate per lane in an SDS-PAGE mini gel. 2) Run at 20 mA per gel until the dye front is close to the bottom. 3) Transfer the proteins to a nitrocellulose membrane (S&S NCTM) at 250 mA in transfer buffer for 1-4 h, depending on the size of the target protein. 4) Incubate the blot with blocking buffer (5% non-fat dry milk in TBS) overnight at 4°C or 2 hr at room temperature (RT). 5) Incubate the blot with primary antibody (diluted 1:250 to 1:1000 in blocking buffer) for 1 hr in blocking buffer at RT. 6) Wash the blot 3 x 10 min in washing buffer (TBS containing 0.1% Tween 20) with shaking. 7) Incubate blot with anti-rabbit IgG-HRP conjugate (Sigma) (diluted 1:10,000 - 1:2,000 in blocking buffer) for 1 h in blocking buffer at RT. 8) Wash 3 x 10 min in washing buffer with shaking. 9) Drain washing buffer, add ECL solution (Amersham) and develop for 1 min. 10) Expose to X-ray film for 1 to 30 min. TBS: • 125 mM NaCl • 25 mM Tris pH 8.0 • 0.1% Tween 20 SDS/Running Buffer: • 25 mM Tris • 192 mM Glycine • 0.1% SDS Transfer Buffer: • 20 mM Tris • 150 mM Glycine • 20% methanol • 0.038% SDS

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