Overview

  • Product name
    Anti-Glutaredoxin 2 antibody - C-terminal
    See all Glutaredoxin 2 primary antibodies
  • Description
    Rabbit polyclonal to Glutaredoxin 2 - C-terminal
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, WB, ELISAmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide selected from the C terminal region of human Glutaredoxin 2 conjugated to KLH (NP_932066)

  • Positive control
    • Mouse cerebellum tissue lysates.

Properties

Applications

Our Abpromise guarantee covers the use of ab85267 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
ICC/IF Use a concentration of 5 µg/ml.
WB 1/50 - 1/100. Detects a band of approximately 18 kDa (predicted molecular weight: 18 kDa).
ELISA 1/1000.

Target

  • Function
    Glutathione-dependent oxidoreductase that facilitates the maintenance of mitochondrial redox homeostasis upon induction of apoptosis by oxidative stress. Involved in response to hydrogen peroxide and regulation of apoptosis caused by oxidative stress. Acts as a very efficient catalyst of monothiol reactions because of its high affinity for protein glutathione-mixed disulfides. Can receive electrons not only from glutathione (GSH), but also from thioredoxin reductase supporting both monothiol and dithiol reactions. Efficiently catalyzes both glutathionylation and deglutathionylation of mitochondrial complex I, which in turn regulates the superoxide production by the complex. Overexpression decreases the susceptibility to apoptosis and prevents loss of cardiolipin and cytochrome c release.
  • Tissue specificity
    Widely expressed. Expressed in brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta and lung. Not expressed in peripheral blood leukocytes.
  • Sequence similarities
    Belongs to the glutaredoxin family.
    Contains 1 glutaredoxin domain.
  • Cellular localization
    Mitochondrion and Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • bA101E13.1 (GRX2 glutaredoxin (thioltransferase) 2) antibody
    • bA101E13.1 antibody
    • CGI133 antibody
    • Glrx2 antibody
    • GLRX2_HUMAN antibody
    • Glutaredoxin-2 antibody
    • Glutaredoxin-2, mitochondrial antibody
    • GRX2 antibody
    • mitochondrial antibody
    see all

Images

  • Anti-Glutaredoxin 2 antibody - C-terminal (ab85267) at 1/50 dilution + mouse cerebellum tissue lysates at 35 µg

    Predicted band size: 18 kDa
    Observed band size: 25 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 40 kDa. We are unsure as to the identity of these extra bands.

  • ICC/IF image of ab85267 stained HeLa 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 (ab85267, 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.

References

This product has been referenced in:
  • Nóbrega-Pereira S  et al. G6PD protects from oxidative damage and improves healthspan in mice. Nat Commun 7:10894 (2016). WB . Read more (PubMed: 26976705) »
  • Wu H  et al. Glutaredoxin 2 (Grx2) gene deletion induces early onset of age-dependent cataracts in mice. J Biol Chem 289:36125-39 (2014). WB ; Mouse . Read more (PubMed: 25362663) »
See all 4 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Answer

Thank you for your response.

Unfortunately, the two attached images have not gone through successfully so I am unable to open them. It is reassuring that Gelsolin and Beta-actin antibodies provide clear signal but they are not positive control since they recognize different targets.

As I have commented on my previous e-mail the expected band for the Glutaredoxin 2 is around 25 kDa. As the datasheet suggests this antibody is not affinity purified (but ammonium sulphate precipitation) and is indeed polyclonal so it recognises different epitopes and it may well be that the 40 kDa band is non-specific. Polyclonal antibodies very often detect shared sequences of similar/related protein families or isoforms(GSH-related enzymes).

Let me know if you have any further questions and how you wish to proceed with this enquiry.

Read More

Question

Dear Sir / Madam,
The customer has replied with the following:



1.Samples:Could you please specify the species and the cell/tissue type used as samples?


-Sample used here are lysates from human colorectal cancer cell line HCT116.


2.Sample preparation:What lysis buffer was applied for preparing the lysate. Could you please provide some details of the compositions?


- I use RIPA buffer from Sigma (ready to use). ( I have used several other lysis buffers as well before for many other Western blots, all with good and clean results).


3.Detection:Does the detection system work fine? Have you used it successfully with another primary antibody?


- Yes, the detection system works perfectly fine (Super Signal West Dura Substrate from Pierce) and it works successfully with all the other primary antibodies I have used so far (refer to attached picture). This is the first primary antibody that I have faced problem with.


4.Incubation:How long and at what temperature was the incubation carried out with the primary antibody? This information is not provided and would be very useful to know.


- Primary antibody was incubated overnight in cold room (4 degrees).


5.Blocking:What blocking solution was applied and How long and at what temperature?


-I have used both 5%BSA and 5% not-fat milk (Blotting-grade blocker from Bio Rad) to block the membrane. The membrane used here is a PVDF membrane (immobilin-millipore, cat no.IPVH00010, pore size-0.45um), that generally does not require blocking once we leave the membrane dry after transfer (1-2 hrs in room temp to dry or quick dry with methanol), and we can just incubate with primary antibody. However, if do not dry the membrane, we can immediately block it while it is still wet. I have tried all these conditions.

Additionally, she has provided another image of a blot following the same technique used for the glrx2 Ab.

Please advise on how we may proceed!


Many Thanks and Best Regards,

Read More
Answer

Thank you very much for getting back to me and for providing some further important details on the experiments. Your co-operation and effort is very much appreciated.

I can confirm that the expected band for the Glutaredoxin 2 is around 25 kDa. As the datasheet suggests this antibody is not affinity purified (but ammonium sulphate precipitation) and is indeed polyclonal so it recognises different epitopes and it may well be that the 40 kDa band is non-specific. Polyclonal antibodies very often detect shared sequences of similar/related protein families or isoforms(GSH-related enzymes).

It is very likely that the band between 25 and 35 kDa in the mitochondrial fraction represents the Glutaredoxin 2.

If you need any further assistance, please do not hesitate to contact me.

Read More

Answer

Thank you for your enquiry regarding ab85267 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this antibody.

Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.

1. Samples: Could you please specify the species and the cell/tissue type used as samples?

2. Sample preparation: What lysis buffer was applied for preparing the lysate. Could you please provide some details of the compositions?

3. Detection: Does the detection system work fine? Have you used it successfully with another primary antibody?

4. Incubation: How long and at what temperature was the incubation carried out with the primary antibody? This information is not provided and would be very useful to know.

5. Blocking: What blocking solution was applied and How long and at what temperature?

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

Read More

Answer

Thank you for sending me your data and details of your protocol.  As we discussed, it is probable that the lowest band is the Glutaredoxin 2 protein, but I have no information as to why the band would appear in one sample and not the other.  You may be able to increase your signal by incubating the primary antibody overnight if you are not already doing so, and by increasing the concentration of your secondary antibody.  Because we have not validated this antibody for use on pig samples, using a positive control such as mouse cerebellum tissue lysates may help to clarify your results.  I hope this helps, please let me know if you have any additional questions or concerns. 

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