Overview

  • Product name
  • Description
    Rabbit polyclonal to SPHK1
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ELISA, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    KLH conjugated synthetic peptide selected from the sequence of human SPHK1 within aa 1-100.

  • Positive control
    • ICC/IF: HepG2 cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab37980 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
ELISA 1/1000.
WB 1/100 - 1/500. Detects a band of approximately 42 kDa (predicted molecular weight: 42 kDa).
ICC/IF Use a concentration of 5 µg/ml.

Target

  • Function
    Catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (SPP), a lipid mediator with both intra-and extracellular functions. Also acts on D-erythro-sphingosine and to a lesser extent sphinganine, but not other lipids, such as D,L-threo-dihydrosphingosine, N,N-dimethylsphingosine, diacylglycerol, ceramide, or phosphatidylinositol.
  • Tissue specificity
    Widely expressed with highest levels in adult liver, kidney, heart and skeletal muscle.
  • Sequence similarities
    Contains 1 DAGKc domain.
  • Information by UniProt
  • Database links
  • Alternative names
    • SK 1 antibody
    • SK1 antibody
    • Sphingosine kinase 1 antibody
    • SPHK 1 antibody
    • SPHK antibody
    • Sphk1 antibody
    • SPHK1_HUMAN antibody
    • SPK antibody
    • SPK 1 antibody
    see all

Images

  • ICC/IF image of ab37980 stained HepG2 cells. The cells were 4% Formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab37980, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 Goat anti-Rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Lane 1 : c-myc tagged SPHK1 antibody
    Lane 2 : Anti-SPHK1 antibody (ab37980) at 0.25 mg/ml

    All lanes : transfected 293 cell lysate

    Predicted band size: 42 kDa
    Observed band size: 47 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 200 kDa. We are unsure as to the identity of these extra bands.

References

This product has been referenced in:
  • Usatyuk PV  et al. Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways. Am J Physiol Lung Cell Mol Physiol 300:L840-50 (2011). WB ; Human . Read more (PubMed: 21478254) »
See 1 Publication for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Answer

Thank you for your email.

Yes, I meant His or GST tag etc.

It is more likely that the band appeared in SPHK2 blot is the specific one. However it can be further testified by
- trying no primary control.
- blocking the antibody with a blocking peptide.
- trying another primary antibody against same target.
- using the isoform specific antibodies.

I hope this information will be helpful. Should you have any other question please do not hesitate to contact me.

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Answer

Thank you for your email.

It is true that the emails are redirected to us if you reply to mailto:technical@abcam.com. I would advise you to log in into your account and check the CCE thread this is the easiest way for tracking the conversation.

I have now received a reply from laboratory. They confirmed doing WB using general western blot protocol at recommended 1/100-1/500 dilutions so this answer isn't very helpful.

The one reason of high molecular weight of SPHK1 is the over-expression of protein with a tag. As you are predicting the band to be nearly 55kDa so I am assuming that the protein is not tagged. If yes, then you might need to add the molecular weight of the tag. I also assume that the conditions were reducing and the samples were properly reduced so possibility of 75kDa as dimer can be excluded.

I am sorry I am unable to determine the exact reason of difference in observed vs predicted band size. You may need to study literature for possible explanation.

For SPHK1 positive control please click the link below;

http://www.proteinatlas.org/ENSG00000176170

SPHK2 is naturally expressed in Mouse NIH3T3 cells, mouse cerebellum, kidney tissues, human colon cancer cells (http://www.sciencedirect.com/science/article/pii/S0014482711004009)

I hope this information will be helpful. Please let us know how we can assist further.

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Answer

Thank you for your email.

I am sorry I might have missed the information about LDS buffer as cell lysis buffer - indeed there are companies who sells these for cell lysis.

I have requested specific protocol from laboratory I will send you this soon. I also have reviewed the images and would like to ask

- What are TM96, 97, 98 and TM91 lysates. Are these cell lines that over expresses SPHK1 and SPHK2?
- Could you please explain why these is no band in HUVEC cell line? The publication http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1533315/ shows positive staining of both targets in HUVEC cells.

Thank you very much for your cooperation. I will further contact you with more information soon.

Read More

Answer

Thank you for contacting us.

Just to confirm I have started working on your inquiry so I will get back to you with more information soon.

There is one comment to make here;

ab37977; This SPHK2 antibody is generated from rabbits immunized with a KLH conjugated synthetic peptide between 598˜627 amino acids from the C-terminal region of human SPHK2. It can potentially recognize all three isoforms (two at approximately 65 and one at 69 kDa).

http://www.uniprot.org/uniprot/Q9NRA0#Q9NRA0

Depending on what type of samples to be tested, the three isoforms can be present and detected either individually or in a combination.

Looking forward receiving an image soon.

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