Overview

  • Product name
    Anti-Cystathionase antibody
    See all Cystathionase primary antibodies
  • Description
    Mouse monoclonal to Cystathionase
  • Host species
    Mouse
  • Tested applications
    Suitable for: WB, IHC-P, ICC/IF, Flow Cytmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Recombinant full length protein, corresponding to amino acids 1-406 of Human Cystathionase

Properties

Applications

Our Abpromise guarantee covers the use of ab54573 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 Use a concentration of 1 - 5 µg/ml. Predicted molecular weight: 45 kDa.
IHC-P Use a concentration of 3 µg/ml.
ICC/IF Use a concentration of 5 µg/ml.
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

Target

  • Function
    Catalyzes the last step in the transsulfuration pathway from methionine to cysteine. Has broad substrate specificity. Converts cystathionine to cysteine, ammonia and 2-oxobutanoate. Converts two cysteine molecules to lanthionine and hydrogen sulfide. Can also accept homocysteine as substrate. Specificity depends on the levels of the endogenous substrates. Generates the endogenous signaling molecule hydrogen sulfide (H2S), and so contributes to the regulation of blood pressure.
  • Pathway
    Amino-acid biosynthesis; L-cysteine biosynthesis; L-cysteine from L-homocysteine and L-serine: step 2/2.
  • Involvement in disease
    Defects in CTH are the cause of cystathioninuria (CSTNU) [MIM:219500]. It is an autosomal recessive phenotype characterized by abnormal accumulation of plasma cystathionine, leading to increased urinary excretion.
  • Sequence similarities
    Belongs to the trans-sulfuration enzymes family.
  • Post-translational
    modifications
    Phosphorylated upon DNA damage, probably by ATM or ATR.
  • Cellular localization
    Cytoplasm.
  • Information by UniProt
  • Database links
  • Alternative names
    • CGL_HUMAN antibody
    • CTH antibody
    • cystathionase (cystathionine gamma-lyase) antibody
    • Cystathionine gamma lyase antibody
    • Cystathionine gamma-lyase antibody
    • Cysteine desulfhydrase antibody
    • Gamma cystathionase antibody
    • Gamma-cystathionase antibody
    • Homoserine deaminase antibody
    • Homoserine dehydratase antibody
    • MGC9471 antibody
    see all

Images

  • Cystathionase antibody (ab54573) at 1ug/lane + K-562 cell lysate at 25ug/lane.
  • Cystathionase antibody (ab54573) used in immunohistochemistry at 3ug/ml on formalin fixed and paraffin embedded human colon.
  • ICC/IF image of ab54573 stained MCF7 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 (ab54573, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight® 488 goat anti-mouse 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.

  • Overlay histogram showing K562 cells stained with ab54573 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab54573, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in K562 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

References

This product has been referenced in:
  • Peh MT  et al. Effect of feeding a high fat diet on hydrogen sulfide (H2S) metabolism in the mouse. Nitric Oxide N/A:N/A (2014). WB ; Mouse . Read more (PubMed: 24637018) »
  • Krishnan N  et al. H2S-Induced sulfhydration of the phosphatase PTP1B and its role in the endoplasmic reticulum stress response. Sci Signal 4:ra86 (2011). WB ; Human . Read more (PubMed: 22169477) »
See all 3 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Answer

Thank you for contacting us.

We do have full length human Cystathionase in our catalog as ab114582.

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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Answer

Thank you for contacting Abcam regarding expiration dates of our products.

At Abcam, we guarantee the effectiveness of our products for a period of 6 months from date of delivery. Should you experience any issues using one of our products in the manner outlined on our datasheets during this time we offer scientific support, replacement or refund.

I did search for the lot number that you provided and found that a product with this lot number was purchased on 22 January 2011 for UNM. While no longer under our Abpromise, this product should remain stable for up to 5 years if upon delivery it was aliquoted and stored at -20°C or -80°C, taking care to avoid repeated freeze / thaw cycles.

I would like to encourage you to take advantage of our Abreview program. It takes just a few minutes to leave a review and you can collect Abpoints which you may redeem for Abcam products or Amazon gift cards while at the same time sharing information about the product with your colleagues worldwide. To leave and Abreview, go to the webpage for the product, click on the Abreview Tab right below the shipping information and clickSubmit an Abreview. You can gain more Abpoints by submitting images as well.

More information may be found at the following link:

https://www.abcam.com/index.html?pageconfig=resource&rid=10332

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

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Question

Hello, I made the following changes: - Loaded 30 ug protein per lane (lane labeled HUVEC 1 was prepared using RIPA lysis buffer and lysate was sufficiently concentrated to load 30 ug; lane labeled HUVEC 2 was prepared from a dilute lysate, using a TCA precipitation) - Used phosphatase inhibitors (Phosphatase Inhibitor Cocktail A or Phosphatase Inhibitor Cocktail B from Santa Cruz) in addition to the protease inhibitor cocktail during cell lysis - Used a 12% SDS-PAGE gel, as compared to the NuPAGE Bis-Tris precast gels used previously I obtained the following results: Blot 1: monoclonal CTH, with phosphatase inhibitors Used 1:250 ab54573. Observed two strong bands that may correspond to the appropriate MW of the two cystathionase variants (see attached). Blot 2: monoclonal CTH, without phosphatase inhibitors Used 1:250 ab54573. Appeared similar to Blot 1. Blot 3: polyclonal CTH, with phosphatase inhibitors Used 1:250 ab80643. Observed several strong bands that do not seem to correspond to the desired 39 or 45 kDa MW. Blot 4: polyclonal CTH with HeLa positive control, without phosphatase inhibitors Used 1:250 ab80643. Detected several strong bands, but slightly different compared to Blot 3. Blot 5: monoclonal CTH antibody from Novus/Abnova Used 1:500 Appeared similar to Blot 1 and 2. Thank you for all of the suggestions. I am very satisfied with the performance of the monoclonal antibody (ab54573), but still have not been able to troubleshoot the polyclonal antibody (a80643) successfully. I appreciate your continued assistance! Sincerely,

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Answer

Thank you very much for keeping me up to date with the performance of the antibodies. I am very happy to hear you could obtain satisfactory results from ab54573. On the other hand it is regrettable ab80643 didn’t perform accordingly. I appreciate the time you have spent in the laboratory and understand your concerns. I can suggest you have regrettably received a bad vial of ab80643. As the order for this antibody was placed within the last 6 months, it is still covered by our Abpromise guarantee, and if you wish to, I can send you a free replacement for this antibody (from a different lot) a reimbursement or a Credit Note in compensation. I am very sorry for the inconveniences, and I will be happy to assist you with this problem, and give a quick and efficient solution. Therefore I look forward to hearing from you letting me know how you would like to proceed. Have a nice day.

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Question

Hello, The antibodies that I have been having difficulty with are for detecting cystathionase in HUVECs: ab54573:, XX-XX-XXXX, lot #: XXXX ab80643:, XX-XX-XXXX, lot #: XXXX The pancreatic cells had been reported to have only low amounts of cystathionase, so we instead moved on to HUVECs which are known to produce cystathionase in detectable quantities. Staining the membrane and the gel showed good transfer of proteins to the membrane. When imaging the blot with secondary antibody, I was able to see strong signal from ab54573 but only for a band present at the top of the gel .I tried digesting the protein lysate at both higher and lower temperatures and still observed the same results. I have not tried a concentration of this antibody higher than 1:500. I wasn't able to see anything on the membrane with the ab80643, trying both 1:1000 and 1:500 concentrations of antibody .Blocking was done in 5% BSA/wash buffer. Since both these antibodies should detect the same protein, I am having the most difficulty determining why the results are so different (one has strong signal albeit in the wrong position, and the other does not). Are these antibodies sensitive to the phosphorylation of the protein? I will look into trying a phosphatase inhibitor with the lysate preparation. I would like to determine whether this lysate preparation is or is not appropriate for the cystathionase protein. Would it be possible to obtain a positive control to determine whether we can detect the protein? Thank you!

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Answer

Thanks for your reply. We don’t have any evidence of the expression of cystathionase in HUVECs. If you have data to support the presence of the protein in the cell line, but not in high quantities, it would be better loading bigger amounts of sample per lane. Attending to the antibodies datasheets, for ab54573, K-562 cell lysate was used to detect Cystathionase. We do have in our catalogue K562 cell lysates that could be used as positive controls. The link to the search result is: https://www.abcam.com/index.html?pageconfig=searchresults&search=K 562&pt=12&sk=targ&sv=K562&sn=K562&l=1&fViewMore=1 For ab80643 the suggested positive control is Transfected 293T cell lysate. Unfortunately, we have 293T cell lysate in out catalogue, but not the transfected one, that would be the one recommended to work with. HeLa lysate can also be used as a positive control. There are some HeLa lysates available in the catalogue, if you wish to have a look: https://www.abcam.com/index.html?pageconfig=searchresults&search=HeLa&pt=12&sk=app&sv=69&sn=WB&l=2&fViewMore=1 I would encourage you to perform the WB again following the recommendations given (higher protein amount, using phosphatase inhibitors, concentrating the ab dilution as well as increasing the secondary concentration) and maybe using any of these lysates to check whether the problem comes from the antibody or from the protein expression level. In case you can’t notice any improvement, please contact me again and I’ll try to give a solution as soon as possible.

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Answer

Thank you for sending these additional details. If we assume the cystathionase in your samples is intact and not degraded then the antibody is probably the issue. I think your protocol is correct, and the Hela lysate, at least, should be giving a signal. Would you be interested in trying the rabbit polyclonal, ab80643, as a free-of-charge replacement? The only data we have is a blot against transfected 293 cells, but we will offer our guarantee (replacement or credit or refund within 6 months of purchase, after considering the protocol) for reactivity with endogenous cystathionase in human samples, e.g. HeLa. Reactivity with other species is predicted based on homology with the immunogen but has not been tested.

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