Overview

  • Product name
    Anti-Glutathione antibody [D8]
    See all Glutathione primary antibodies
  • Description
    Mouse monoclonal [D8] to Glutathione
  • Host species
    Mouse
  • Specificity
    This antibody reacts with glutathione-protein complexes under non-reducing conditions.
  • Tested applications
    Suitable for: IHC-FoFr, WB, IHC-Fr, IHC-P, Flow Cyt, IP, ELISA, ICC, ICC/IFmore details
  • Immunogen

    Glutathione conjugated to KLH

Properties

Applications

Our Abpromise guarantee covers the use of ab19534 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
IHC-FoFr Use at an assay dependent concentration. PubMed: 19833109
WB 1/1000. Use under non reducing condition. We recommend blocking with 5% milk (not BSA). While glutathione itself is too small to detect in WB, this antibody will detect all glutathionylated proteins. You may observe multiple bands at variable molecular weights depending on what proteins in your samples are glutathionylated.
IHC-Fr 1/100 - 1/200.
IHC-P Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

 

IP Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
ICC Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.

Target

  • Relevance
    Glutathione is a small peptide composed of three amino acids: cysteine, glutamic acid, and glycine and is present in tissues in concentrations as high as one millimolar. It contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side chain. Glutathione is involved in detoxification, it binds to toxins, such as heavy metals, solvents, and pesticides, and transforms them into a form that can be excreted in urine or bile. It is also an important antioxidant, helping to maintain the -SH groups of proteins in their reduced form. Chronic functional glutathione deficiency is associated with glucose 6-phosphate dehydrogenase deficiency, immune disorders, an increased incidence of malignancies, and in the case of HIV disease, probably accelerated pathogenesis of the disease. Acute manifestations of functional glutathione deficiency can be seen in those who have taken an overdosage of acetaminophen (paracetamol). This results in depletion of glutathione in the hepatocytes, leading to liver failure and death.
  • Alternative names
    • Glutathione antibody
    • GSH antibody
    • Oxidised glutathione antibody
    • Reduced glutathione antibody

Images

  • ab19534 staining Glutathione in cultured murine RAW 264.7 cells by Immunocytochemistry/ Immunofluorescence.
    Cells were fixed in paraformaldehyde, permeabilized using 0.1% Triton-X100 in 2% BSA for 15 minutes, blocked with 2% BSA for 1 hour at 22°C and then incubated with ab19534 at a 1/150 dilution for 16 hours at 4°C. The secondary used was an Alexa-Fluor 488 conjugated goat anti-mouse IgG (H+L) used at a 1/1000 dilution.

    See Abreview

  • ab19534 at a 1/250 dilution detecting Glutathione in human monocytes by Flow Cytometry. An Alexa-Fluor 488 conjugated goat anti-mouse IgG (H+L) secondary was used at a 1/500 dilution.

    See Abreview

  • ab19534 staining Glutathione in human neutrophils by Immunocytochemistry/ Immunofluorescence.
    Samples were fixed with 4% (w/v) paraformaldehyde in PBS, permeabilized with PBS containing 0.5% (w/v) saponin and 0.1% (w/v) bovine serum albumin, and then blocked with 1% (w/v) bovine serum albumin in PBS for 1 hour. ab19534 was used at 5µg/ml for 2 hours at room temperature. After washing with PBS, cells were incubated with Alexa 488-conjugated anti-mouse IgG at a 1/200 dilution.
  • ab19534 staining glutathione in A549 cells treated with apocynin (ab120615), by ICC/IF. Increase in glutathione expression correlates with increased concentration of apocynin, as described in literature.
    The cells were incubated at 37°C for 24h in media containing different concentrations of ab120615 (apocynin) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab19534 (10 µg/ml) was performed overnight at 4øC in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-mouse polyclonal antibody (ab96879) at 1/250 dilution was used as the secondary antibody.

References

This product has been referenced in:
  • Dou X  et al. Glutathione disulfide sensitizes hepatocytes to TNFa-mediated cytotoxicity via IKK-ß S-glutathionylation: a potential mechanism underlying non-alcoholic fatty liver disease. Exp Mol Med 50:7 (2018). WB . Read more (PubMed: 29622764) »
  • Seol HS  et al. Glutamate release inhibitor, Riluzole, inhibited proliferation of human hepatocellular carcinoma cells by elevated ROS production. Cancer Lett 382:157-165 (2016). Read more (PubMed: 27612558) »
See all 14 Publications for this product

Customer reviews and Q&As

1-10 of 18 Abreviews or Q&A

Answer




"It is technically challenging and we do not recommend it for quantitative analysis.

The best method developed so far is that of Sun and Kalloniatis, J Comp Neurol 494:686 -703, 2006.

We advise to consider this when doing cryosections immunocytochemistry for small molecules like glutathione:

1. All our small molecule antibodies are designed for post embedding immunocytochemistry using plastic resin. Cryo imaging is not a technology we recommend, but we understand some users wish to use it anyway. It is possible, but the user must appreciate that this turns a quantitative method into a qualitative one.

2. Small molecule detection requires some glutaraldehyde fixation. We recommend 0.05% for about 60 minutes followed by a buffer wash and conventional cryoimmunocytochemistry with standard detergent penetration. The choice of visualization is up to the user, recognizing that glutaraldehyde increases background fluorescence, mostly in the the green (Alexa 488) range. This is less of a problem for Cy3, Cy3.5 and other very red dyes. It is not a problem for HRP/DAB. Typically the 1:100 primary dilution we recommend may be a little strong for cryo and lower levels of 1:200-1:500 may be necessary, depending on how much glutathione there is in the tissue (e.g. kidney and liver have much higher levels than skeletal muscle, heart or nervous system).

3. All cells have some glutathione ranging from 100 uM to 10 mM depending on types. This means that in a superposition image, there will be a very high glutathione "background." Thus we recommend confocal imaging to minimize this superposition effect."

I hope thisadditional information is helpful and wish godd luck with your research.

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Answer

I have been informed by the lab that the conjugation of KLH to glutathione was through the thiol group.

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Answer

As requested I issued a credit note. The credit note ID xxxxxx.

As usual if you have any further questions regarding this credit note, please contact the accounts department by email at creditcontrol@abcam.com. Please refer to the credit ID number in any correspondence with the accounting department.

I would like to wish the customer good luck with their research. The technical team is always at your service, should you require further expert advice.

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Answer

Thank you for your message and for providing this further information.

I am sorry to hear the suggestions made have not improved the results on this occasion.
I appreciate the time your customer has spent on these experiments and would be pleased to arrange a replacement with a different lot number or a credit note in compensation.

I look forward to hearing from you with details of how you and your customer would like to proceed.

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Answer



We have 2 antibodies that react with conjugated glutathione and 2 that are designed to react with free glutathione. I have looked at these products and it doesn't appear any of them have been specifically tested for reactivity to glutathione as a thioether. So they may or may not react with glutathione as a thioether.

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Answer



I'm sorry but at this time we don't have an antibody that reacts specifically to glutathione as a thioether.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Tissue lysate - whole (Liver)
Loading amount
10 µg
Specification
Liver
Treatment
10mM Diamide
Gel Running Conditions
Non-reduced Non-Denaturing (Native)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2.5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Sep 05 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HeLa cells)
Loading amount
35 µg
Specification
HeLa cells
Treatment
Diamide for 24h
Gel Running Conditions
Non-reduced Non-Denaturing (Native)
Blocking step
Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Sep 05 2012

Answer

Vielen Dank für Ihre Anfrage.

Glutathion ist nicht Spezies spezifisch, da es immer aus Cystein, Glutaminsaeure und Glycin besteht.

Wir haben ab19534 nicht speziell mit Rattenproben getestet, gehen aber davon aus, dass der Antikörper Glutathion überall erkennt.

Er ist für IHC-Fr getestet und auch garantiert und damit von unserer Abpromise Garantie abgedeckt, wenn Sie ihn in IHC-Fr auf Rattenproben einsetzen.

Ich hoffe, diese Information ist hilfreich und wünsche Ihnen viel Erfolg bei Ihren Versuchen.

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Answer

Thank you for contacting us.

Glutathione itself will be too small to view in a WB, but the customer will be able to detect any proteins that are glutathionylated in his or her samples. The results will show multiple bands at varying molecular weights depending on which proteins are glutathionylated. As a positive control, this antibody is validated using purified human phophotyrosine phosphatase 1B, which produces a band of ˜42 kDa.

To run a non-reducing WB, beta-mercaptoethanol and DTT should be omitted from the sample buffer. For detailed information about reducing and denaturing conditions, I would recommend that the customer review the sample preparation section of our WBguide:

https://www.abcam.com/index.html?pageconfig=resource&rid=11379#A6

I hope this helps, please let me know if you need any additional information or assistance.

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1-10 of 18 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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