Overview

  • Product name

  • Description

    Rabbit polyclonal to SPHK2
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IP, ELISA, WB, IHC-Pmore details
  • Species reactivity

    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide conjugated to KLH, derived from within residues 600-700 of SPHK2 (Human).

  • Positive control

    • Human cancer tissue, transfected 293 cell lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab37977 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
IP Use at an assay dependent concentration. PubMed: 24659784
ELISA 1/1000.
WB 1/1000. Predicted molecular weight: 69 kDa.
IHC-P 1/50 - 1/100. Perform enzymatic antigen retrieval before commencing with IHC staining protocol.

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-dihydrosphingosine, D-erythro-sphingosine and L-threo-dihydrosphingosine. Binds phosphoinositides.
  • Sequence similarities

    Contains 1 DAGKc domain.
  • Cellular localization

    Cytoplasm. Membrane and Lysosome membrane.
  • Information by UniProt
  • Database links

  • Alternative names

    • SK 2 antibody
    • Sphingosine kinase 2 antibody
    • Sphingosine kinase type 2 antibody
    • SPHK2 antibody
    • SPHK2_HUMAN antibody
    • SPK 2 antibody
    see all

Images

  • Anti-SPHK2 antibody (ab37977) at 1/1000 dilution + HepG2 cell Lysate at 35 µg

    Secondary
    HRP goat anti-rabbit (H+L) at 1/5000 dilution

    Predicted band size: 69 kDa



    Incubation time was overnight at 4°C. Blocking/Dilution buffer: 5% NFDM/TBST.

  • Immunohistochemical staining of SPHK2 in Human breast carcinoma tissue sections (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections) with ab37977 at a dilution of 1/25. Tissue was fixed with formaldehyde and blocked with 3% BSA for 0.5 hours at 38°C. Antigen retrieval was heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody (1/25) for 1 hour at 37°C. A HRP-conjugated goat anti-rabbit polyclonal (ready to use) was used as the secondary antibody.

  • Anti-SPHK2 antibody (ab37977) at 1/500 dilution + Mouse NIH 3T3 whole cell lysate at 25 µg

    Secondary
    An HRP-conjugated Donkey Polyclonal at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 69 kDa



    Blocking Step: 5% Milk for 1 hour at 22°C.

    See Abreview

References

This product has been referenced in:

  • Han C  et al. Sphk1/S1P/S1PR1 Signaling is Involved in the Development of Autoimmune Thyroiditis in Patients and NOD.H-2h4 Mice. Thyroid 29:700-713 (2019). Read more (PubMed: 30963819) »
  • Kreitzburg KM  et al. FTY720 enhances the anti-tumor activity of carboplatin and tamoxifen in a patient-derived xenograft model of ovarian cancer. Cancer Lett 436:75-86 (2018). Read more (PubMed: 30120964) »
See all 21 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - whole cell (HepG2)
Gel Running Conditions
Reduced Denaturing
Loading amount
25 µg
Treatment
100 ng/ml LPS
Specification
HepG2
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Nov 19 2018

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.

Read More

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.

Read More

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.

Read More

Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee for WB and in mouse and human samples. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

This SPHK2 antibody can potentially recognize all three isoforms of the protein (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. So this could give different band sizes on the blot.

We have not tested cross-reactivity with SPHK1,but the immunogen sequence does not share significant homology with SPHK1.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you could complete this.
It will help you put the information we require together very easily.

I would appreciate if you are also able to provide an image which would help us to assess the results.

Thank you for your time and cooperation. We look forward to receiving the completed questionnaire.



Order Details
Antibody code:


Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background

Lot number

Purchase order number
or preferably Abcam order number:



General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, wrong band size, more bands, no band etc.)


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


Amount of protein loaded


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method (ECL, ECLPlus etc.)


Positive and negative controls used (please specify)



Optimization attempts (problem solving)
How many times have you tried the Western?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?


What steps have you altered?


Additional Notes:


Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to assess the results.

Read More

Answer

Merci de nous avoir contactés.

Nous sommes désolés d'apprendre que le produit que vous avez reçu ne fonctionne pas comme attendu. Le numéro de commande de remplacement gratuit avec la référence ab37977est : *****.

Vous recevrez prochainement un mail de confirmation comprenant les détails d'expédition.

N'hésitez pas à nous contacter lors d'une prochaine occasion.

Read More

Question
Answer

Thank you for your call earlier this week and for your patience. The blocking peptide of ab37977 is now available on the catalog as ab115809- https://www.abcam.com/SPHK2-peptide-ab115809.html Please let me know if you have any questions or if there is anything else that we can do for you.

Read More
Application
Western blot
Sample
Mouse Cell lysate - whole cell (NIH 3T3)
Loading amount
25 µg
Specification
NIH 3T3
Treatment
hypoxia
Gel Running Conditions
Reduced Denaturing (10%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Feb 22 2010

Application
Western blot
Sample
Human Cell lysate - whole cell (HMEC endothelial cells)
Loading amount
25 µg
Specification
HMEC endothelial cells
Treatment
hypoxia
Gel Running Conditions
Reduced Denaturing (10%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted Feb 22 2010

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