Overview

  • Product name
  • Description
    Rabbit polyclonal to Glutathione
  • Host species
    Rabbit
  • Specificity
    Because these IgGs were specifically designed to detect glutaraldehyde-linked haptens rather than free GSH or GSSG, the only affinity data is based on IC50 values for artificial antigens constructed as J-GA-J reagents, where J = GSH or GSSG and GA = glutaraldehyde. No measurable glutaraldehyde-fixed tissue cross-reactivity (<1:1000) was detected against L-alanine, gamma-aminobutyrate, D/L-aspartate, agmatine, guanidine, D/L-arginine, L-citrulline, L-cysteine, D/L-glutamate, D/L-glutamine, glycine, L-lysine, L-ornithine, L-serine, taurine, L-threonine, L-tryptophan, L-tyrosine. The antibody does not discriminate between free GSH and GSSG nor does it discriminate between oxidised and reduced GSH.It detects total "free" glutathione.
  • Tested applications
    Suitable for: IHC-FoFr, IHC-Fr, ICC, Immunomicroscopymore details
  • Immunogen

    Chemical/ Small Molecule by a Glutaraldehyde linker.

Properties

Applications

Our Abpromise guarantee covers the use of ab9443 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.
IHC-Fr Use at an assay dependent concentration.
ICC Use at an assay dependent concentration.
Immunomicroscopy Use at an assay dependent concentration. This antibody is recommended for thin section post-embedding endogenous content mapping by Light and Electron microscopy immunostaining via High Performance Immunocytochemistry (HPI) (see "Protocols" tab above).

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

References

ab9443 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Question
Answer

I have been in touch with the lab regarding the concentration of this product, ab9443 anti-glutathione polyclonal. This IgG is prepared as an IgG fraction from serum. It is not an affinity purified pure anti-glutathione IgG so the lab does not have protein concentration. The estimated total IgG fraction in the stock vial is about 125 ug/ml.

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Answer


ab9443 is a product designed for immunocytochemistry

1. ab9443 is an anti-glutathione polyclonal IgG prepared as an IgG fraction from serum. It is not an affinity purified pure anti-glutathione IgG. Thus it does not have protein concentration.

2. The estimated total IgG fraction in the stock vial is about 125 ug/ml.

3. ab9443 is calibrated for use in immunocytochemistry at a dilution of 1:100.

4. Detecting glutathionylation in proteins.
ab9443 is designed for immunocytochemistry on glutaraldehyde fixed specimens, not for Westerns.
It is engineered to detect glutathione fixed to tissue proteins by bifunctional glutaraldehyde - with one aldehyde binding to tissue protein and the other to the alpha amino group of the gamma-linked glutamate residue in free cytoplasmic glutathione.
a. The functional epitope includes the entire glutaraldehyde - glutamate - cysteine - glycine backbone.
b. The functional epitope does NOT include the -SH face of the cysteine residue.
c. It will detect free glutathione (both GSH adn GSSG), but with an affinity ≈ 100x lower than fixed glutathione
d. It can detect highly glutathionylated, abundant proteins in Western blots; however, these epitopes lack the glutaraldehyde link, the efficacy will be much lower than the ability to detect free "fixed" glutathione in sections.
While ab9443 can be used for this purpose, it was not designed for it.

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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

ab9443 is an anti-Glutathione polyclonal IgG prepared as an IgG fraction from serum. It is not an affinity purified pure anti-glutathione IgG thus we don't have a pure protein concentration for it.

As a very rough estimate, we believe that the concentration will be in the region of 1-10mg/ml.

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Question
Answer

Thank you for your enquiry. The following publication shows glutathione (GSH) expression in frozen sections from various tissues including liver, lung, kidney, testis and eye (Fig. 3 & 4). HISTOCHEMICAL LOCALIZATION OF GLUTATHIONE IN TISSUES. THE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY. Vol. 23, No. 10. pp. 774-779, 1975. http://www.jhc.org/cgi/reprint/23/10/774 Although the application used was for frozen sections, the tissue types may also serve as a good positive control for IHC-P. I hope this information will be helpful. If there is anything else that I can help you with, please do not hesitate to contact me.

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Question
Answer

Thank you for your enquiry. These are the references we have for ab9443: Joyce-Brady M, RE Marc, JC Jean 2002 The importance of gamma-Glutamyl transferase in lung glutathione homeostasis and antioxidant defense in Thiol Metabolism and Redox Regulation of Cellular Functions (Pompella et al. Eds) IOS Press, 182-196. Jean JC, Y Liu, LA Brown, RE Marc, E Klings, M Joyce-Brady 2002 Gamma- glutamyltransferase deficiency results in lung oxidant stress in normoxia. Am J Physiol Lung Cell Mol Physiol 283: 766-776. Marc RE and D Cameron 2002 A molecular phenotype atlas of the zebrafish retina. J Neurocytol. 30: 593-654. Regarding IHC on frozen sections, our source for ab9443 replies that they have no validated protocols using frozen sections and explicitly do not recommend or support that method. For technique, please consult the following references: Hjelle OP, Chaudhry FA, Ottersen OP. Antisera to glutathione: characterization and immunocytochemical application to the rat cerebellum. Eur J Neurosci. 1994 May 1;6(5):793-804. Pow DV, Crook DK. Immunocytochemical evidence for the presence of high levels of reduced glutathione in radial glial cells and horizontal cells in the rabbit retina. Neurosci Lett. 1995 Jun 23;193(1):25-8. Marc RE and D Cameron 2002 A molecular phenotype atlas of the zebrafish retina. J Neurocytol. 30: 593-654. These all use the optimal methods for glutathione detection. If you have any other questions, please do not hesitate to contact us.

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Answer

Thank you for your enquity. At the moment we have 2 glutathione antibodies, ab9443 and ab8883, neither of which have yet been tested for application in Western blotting. To see if another source may have what you are looking for, you can click on the link to "The World's Antibody Gateway". The World's Antibody Gateway is a free search engine service provided by Abcam to help you to quickly find the antibodies that you are looking for. It is a free-text search engine developed by the Abcam team so that it searches the catalogues of all online antibody companies (currently 249).

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Question
Answer

Yes. Antibodies against small molecule haptens (e.g. GTH) work in all species, including plants. As with all free small molecule targets, the fixative must include glutaraldehyde and the best visualization results obtained with silver intensification on resin sections. A reference for the use of anti-glutathione in mouse lung is: Jean JC, Y Liu, LA Brown, RE Marc, E Klings, M Joyce-Brady 2002 Gamma-glutamyltransferase deficiency results in lung oxidant stress in normoxia. Am J Physiol Lung Cell Mol Physiol 283: 766-776.

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Answer

The antibody with which you are currently working (ab8883) has only been tested in ELISA. ab9443 has been tested in many IHC-related applications (Formalin fixed sections, Immunohistochemistry (Frozen Sections), Immunocytochemistry, Immunomicroscopy) so I would certainly recommend it. As yet, we are not aware of any publications featuring the use of this antibody.

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

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