Overview

  • Product name
  • Description
    Rabbit polyclonal to Glutaminase
  • Host species
    Rabbit
  • Tested applications
    Suitable for: Flow Cyt, WB, ICC/IF, IHC-Fr, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human Glutaminase aa 516-545 (C terminal) conjugated to Keyhole Limpet Haemocyanin (KLH).

  • Positive control
    • WB: 293, 239T and HeLa cell lysates, mouse liver and brain tissue lysates, human brain and rat kidney tissue lysates. ICC/IF: HeLa and HepG2 cells. IHC-P: Human liver and mouse brain tissues. Flow Cyt: HepG2 cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab93434 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
Flow Cyt 1/10 - 1/50.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

 

WB 1/1000. Predicted molecular weight: 65 kDa.
ICC/IF Use a concentration of 5 µg/ml.
IHC-Fr Use at an assay dependent concentration.
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • Function
    Catalyzes the first reaction in the primary pathway for the renal catabolism of glutamine.
  • Tissue specificity
    KGA is expressed predominantly in brain and kidney but not in liver, isoform 3 is expressed principally in cardiac muscle and pancreas but not in liver or brain, and isoform 2 is expressed solely in cardiac and skeletal muscle.
  • Sequence similarities
    Belongs to the glutaminase family.
    Contains 1 ANK repeat.
  • Cellular localization
    Mitochondrion.
  • Information by UniProt
  • Database links
  • Alternative names
    • AAD20 antibody
    • DKFZp686O15119 antibody
    • FLJ10358 antibody
    • GAC antibody
    • GAM antibody
    • GLS antibody
    • GLS1 antibody
    • GLSK_HUMAN antibody
    • Glutaminase C antibody
    • Glutaminase kidney isoform antibody
    • Glutaminase phosphate activated antibody
    • K-glutaminase antibody
    • KGA antibody
    • KIAA0838 antibody
    • L-glutamine amidohydrolase antibody
    • mitochondrial antibody
    see all

Images

  • Anti-Glutaminase antibody (ab93434) at 1/1000 dilution + Rat kidney tissue lyate at 35 µg

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

    Predicted band size: 65 kDa

  • IHC image of ab93434 staining in human liver formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab93434, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • ICC/IF image of ab93434 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 (ab93434, 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.

  • Flow cytometry analysis of HepG2 cells labelling Glutaminase (green) with ab93434 compared to a negative control (blue). A FITC-conjugated goat anti-rabbit IgG was used as the secondary antibody.

  • All lanes : Anti-Glutaminase antibody (ab93434) at 1/1000 dilution

    Lane 1 : 293 cell lysate
    Lane 2 : 293T cell lysate
    Lane 3 : HeLa cell lysate
    Lane 4 : Human brain tissue lysate
    Lane 5 : Mouse brain tissue lysate

    Lysates/proteins at 35 µg per lane.

    Secondary
    All lanes : HRP-conjugated goat anti-rabbit IgG (H+L) at 1/5000 dilution

    Predicted band size: 65 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse brain tissue labelling Glutaminase with ab93434. A peroxidase-conjugated anti-rabbit IgG was used as the secondary antibody.

  • Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Glutaminase (green) with ab93434. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG was used as the secondary antibody. Counter stained with DAPI (blue).

  • Anti-Glutaminase antibody (ab93434) at 1/1000 dilution + mouse liver tissue lysate at 35 µg

    Secondary
    Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/5000 dilution

    Predicted band size: 65 kDa



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

References

This product has been referenced in:
  • Jiang S  et al. Let-7 Suppresses B Cell Activation through Restricting the Availability of Necessary Nutrients. Cell Metab 27:393-403.e4 (2018). Read more (PubMed: 29337138) »
  • Mateus I  et al. Glucose and glutamine handling in the Sertoli cells of transgenic rats overexpressing regucalcin: plasticity towards lactate production. Sci Rep 8:10321 (2018). Read more (PubMed: 29985416) »
See all 22 Publications for this product

Customer reviews and Q&As

1-10 of 16 Abreviews or Q&A

Answer

Thank you for your email.

Unfortunately, the exact sequence of the immunogen for ab93434 is confidential and I was denied access to it.

But I am happy to confirm that ab93434 is generated from rabbits immunized with a KLH conjugated synthetic peptide between 523˜553 amino acids from the C-terminal region of human GLS.

I hope this information is helpful and wish good luck with your experiments.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (NCM460 colon cells)
Gel Running Conditions
Reduced Denaturing (10)
Loading amount
20 µg
Specification
NCM460 colon cells
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Oct 02 2018

Application
Immunohistochemistry (PFA perfusion fixed frozen sections)
Sample
Rat Tissue sections (brain)
Antigen retrieval step
None
Permeabilization
No
Specification
brain
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Sep 14 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Rat Tissue sections (Brain section - cortical neurons)
Specification
Brain section - cortical neurons
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.1% Triton X-100
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 21°C

Dr. Richard Fairless

Verified customer

Submitted Mar 18 2013

Answer

DISCOUNT CODE: ABTRIAL-00000
Expiration date: 18. March 2013
Value: €370.00

I am very pleased to hear you would like to accept our offer and test ab93434 in rat. This code will give you €370.00 off your next order before the expiration date. To redeem this offer, please submit an Abreview for rat and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.


Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways:
1) Call to place your order and mention the code to our customer service department;
2) Include the code in your fax order;
3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/abtrial.

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Answer

Thank you very much for your interest our products.

I am happy to confirm that the immunogen of ab93434 shares a 90% homology with rat and therefore it is likely that ab93434 cross reacts with rat.


To our knowledge, ab93434 has not been experimentally tested in rat. However by participating in our AbTrial program you can now use our products in an untested application or species without financial risk.

Simply follow these easy steps below to apply for our AbTrial Program:

1. Reply to this email, letting us know you are interested in testing this product.

2. Our scientists will email you an inactive personal discount code for the value of the product.

3. Purchase and test the product at the regular price.

4. Submit your results, including your discount code in the additional notes section of your Abreview.

5. Once the Abreview is submitted, the discount code will become active.

6. Apply your discount code on your next order to receive that value off.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/abtrial.

Read More

Answer

Thank you for contacting us.

The antibody is raised against an amino acid sequence derived from the kidney isoform, product of the GLS gene, but the sequence shares 87% identity with the product of the GLS2 gene, the liver isoform. This probably accounts for the cross reaction with the liver samples.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Answer

Thank you for your inquiry.

This antibody is for Glutaminase kidney isoform, mitochondrial. The antibody will recognize isoforms 1 & 3.

http://www.uniprot.org/uniprot/O94925

Out tested positive control in WB is mouse liver tissue lysate. The immunogen of this antibody is from amino acid 523 - 553 from C-terminal end of human GLS (kidney) protein. This sequence shares 87% homology with liver GLS protein so theoretically this antibody will detect both kidney and liver isoforms.

The MW in WB may be different between the antibodies because different immunogens were used so different isoforms will be detected, and different samples were tested which may express different isoforms.

The correct MW should be between 65-73 kDa.

I hope this information helps. Please contact us with any other questions.

Read More

Answer

Thank you for your enquiry.

I am sorry to confirm that ab93434 and ab60709 have not been specifically tested on brain tissue in western blot and so regrettably there is no data to provide.
I would like to reassure you that ab93434 and ab60709 are both tested and covered by our 6 month guarantee in Western blot and human samples (this would include brain samples). If the antibody does not work as stated on the datasheet, we can provide a refund, free of charge replacement or credit.

For more information regarding the guarantee:
https://www.abcam.com/index.html?pageconfig=abpromise

With regards to publications, we have the following listed on the datasheets which I hope will be helpful.Any publications that we are aware of will be listed on the datasheet. We have a dedicated team who spend time reviewing the literature to find articles that have used our products. However, in many cases, regrettably we do not have access to all the articles mentioning our products, or subscriptions to all the journals that publish them:

ab60709 Anti-Glutaminase antibody

Kung HN et al. Glutamine synthetase is a genetic determinant of cell type-specific glutamine independence in breast epithelia. PLoS Genet 7:e1002229 (2011). WB; Human.Read more (PubMed: 21852960)

Chambers JW et al. Glutamine metabolism is essential for human cytomegalovirus infection. J Virol 84:1867-73 (2010). WB; Human.Read more (PubMed: 19939921)

Premature senescence of human endothelial cells induced by inhibition of glutaminase.
Unterluggauer H, Mazurek S, Lener B, Hutter E, Eigenbrodt E, Zwerschke W, Jansen-Durr P.Biogerontology. 2008 Aug;9(4):247-59. Epub 2008 Mar 4.

ab93434Glutaminase antibody

Colombo SL et al. Molecular basis for the differential use of glucose and glutamine in cell proliferation as revealed by synchronized HeLa cells. Proc Natl Acad Sci U S A 108:21069-74 (2011). WB; Human.Read more (PubMed: 22106309)

Should you have any further questions, please do not hesitate to contact me.

Read More

Answer

Thank you for providing me with this extra information. It has allowed me to more fully understand the problems you have been experiencing with ab49873 and ab93434 previously.

Having looked at the protocol I have a few further questions:

1. Exactly how was the tissue fixed? For how long was the tissue treated with PFA? What concentration of PFA was used?

2. What were the samples tested? Which species did the tissue come from?

3. Had the goat anti rabbit used for both of the experiments been used successfully before?

As ab93434 has as yet to our knowledge not been used in staining frozen sections it may be that the antibody is not suitable for this application. However, there are a few suggestions I can make which may help toimprove the results seen so far.

1. I would suggest decreasing the dilution of both the antibodies. Although ab49873 is recommended to be used at 1/5000, this is only a starting point and may need to be optimised depending on the tissue and protocol used. With ab49873, I would suggest initially trying 1/500 to see if there is any signal. With ab93434 we recommend using 5 µg/ml as an initial starting point, I would therefore use a dilution of 1/50 to start.

2. As you have not been observing any staining with overnight incubation at 4 degrees, I would try incubating at room temperature for 2 hours to see if this improves the results.

3. I would increase the level of Triton X used to 0.2 % for performing the permiabilisation.

I hope these protocol tips are of use, with the answers to the questions above I may be able to provide further suggestions.

I am able to send you ab91073 directly as I suggested to detect theL isoform of glutaminase as a good-will gesture for the bad experience you have had. If you would prefer to change this to ab60709 I can also organise this.

I look forward to receiving your reply.

Read More

1-10 of 16 Abreviews or Q&A

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