Overview

  • Product name
    GST 6XHis-tag ELISA Kit
  • Detection method
    Colorimetric
  • Precision
    Intra-assay
    Sample n Mean SD CV%
    Overall 8 6.4%
    Inter-assay
    Sample n Mean SD CV%
    Overall 3 10%
  • Sample type
    Cell Lysate
  • Assay type
    Sandwich (quantitative)
  • Sensitivity
    > 0.5 ng/ml
  • Recovery

    Sample specific recovery
    Sample type Average % Range
    Serum 75 66% - 90%
    Cell culture extracts 94 66% - 112%
    Cell culture media 90.1 78% - 106%

  • Assay duration
    Multiple steps standard assay
  • Product overview

    The 10X blocking buffer is casein based. It is normal for it to be cloudy with small precipitates in it. This does not negatively affect the assay.


    The 6XHis-tag is a commonly used polyhistidine tag for the purification of recombinant proteins by immobilized metal ion affinity chromatography (IMAC) from E. coli, Saccharomyces cerevisiae, mammalian cells and baculovirus-infected Spodoptera frugiperda insect cells. It is typically used (1) as a single tag on proteins for purification, (2) as part of a dual tag for achieving proteins of higher purity (i.e. combined with GST fusion partner, albumin-binding protein or S-peptide) or (3) in tandem affinity purification of endogenous protein complexes.


    This 6XHis-tag detection assay kit is an enzyme immunoassay developed for the sensitive detection of 6XHis-tagged fusion proteins with a suitable detector antibody or for the quantification of GST-6xHis double tagged proteins with the supplied detector antibody. The quantity of the GST 6XHis double tag can be determined in lysates or intermediate purification fractions by comparing their absorbance with that of a known recombinant GST 6XHis-tagged standard curve. The kit has a detection sensitivity limit of 0.5ng/mL of GST 6XHis-tagged protein. The kit is particularly suitable for screening in high-throughput conditions for maximum expression and optimal purification of 6XHis-tagged proteins as well as for screening humoral immunity against His-tagged antigens. Each kit provides sufficient reagents to perform up to 96 assays including standard curve and 6XHis-tag fusion proteins.


    This kit is reactive with any 6XHis-tagged recombinant protein.

  • Notes

    Typical working ranges:

    GST His tagged: 1 – 50 ng/mL

    GSK3b His tagged: 70 – 5000 ng/mL

    The 10X blocking buffer is casein based. It is normal for it to be cloudy with small precipitates in it. This does not negatively affect the assay. 

  • Tested applications
    Suitable for: Sandwich ELISAmore details
  • Platform
    Microplate

Properties

  • Storage instructions
    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    10X Blocking Buffer 1 x 6ml
    10X GST Detector Antibody 1 x 700µl
    10X HRP Label 1 x 1ml
    20X Buffer 1 x 20ml
    Anti 6XHis-tag Microplate (8 x 12 antibody coated strips) 1 unit
    Extraction Buffer 1 x 15ml
    GST Standard 1 x 2µg
    HRP Development Solution 1 x 12ml
  • Research areas
  • Relevance
    The H-H-H-H-H-H motif is used as a tag on many recombinant proteins to facilitate purification. The antibody recognizes the His-tag fused to the amino- or carboxy- termini of targeted proteins in transfected or transformed cells.
  • Cellular localization
    Depends upon the localization of the parent protein tagged with hexahistidine.
  • Alternative names
    • 6 His epitope tag
    • Hexa His tag
    • HHHHHH epitope tag
    • HHHHHH tag
    • His tag
    • Polyhistidine Tag
    see all

Applications

Our Abpromise guarantee covers the use of ab128573 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
Sandwich ELISA Use at an assay dependent concentration.

Images

  • This kit is effective with a wide variety of sample matrices.
  • Validation by Western blot. Capture antibody was tested by western blot against three His tagged proteins: GST recombinant protein loaded at 50ng/well (lane 1), GSK3b recombinant protein loaded at 50ng/well (lane 2) and Hexokinase 1 recombinant protein loaded at 250ng/well (lane 3). Blots were blocked with 5% Milk in TBST buffer, incubated overnight in anti 6XHis-tag primary antibody and labeled with GAM HRP.
  • Example GSK3B-His tagged standard curve. GSK3B-His tagged protein (not provided with the kit) was diluted from 5µg/mL to 0.07µg/mL and loaded on to the His plate. GSK3b was detected with a rabbit monoclonal antibody and labeled with the provided HRP label. Background was subtracted from data points prior to log transformation and curve fitting (log-agonist vs. response – variable slope).
  • Example GST 6XHis tagged standard curve. GST-His tagged protein (provided with the kit) was diluted from 50 ng/mL to 0.7 ng/mL. Background was subtracted from data points prior to log transformation and curve fitting (log-agonist vs. response – variable slope). Sensitivity was calculated mathematically to be as low as 0.5 ng/mL.

Protocols

References

This product has been referenced in:
  • Ding L  et al. Small sized EGFR1 and HER2 specific bifunctional antibody for targeted cancer therapy. Theranostics 5:378-98 (2015). Read more (PubMed: 25699098) »
See 1 Publication for this product

Customer reviews and Q&As

1-10 of 11 Abreviews or Q&A

Answer

Thank you for contacting Abcam earlier today.

I think I have figured out the protocol for this product. Yes, you can use it with a His-tagged protein, but I think the following sentence from page 3 of the protocol guide clarifies the situation:

"Bound proteins can be detected using rabbit antibody specific to the 6XHistagged fusion protein of choice (not included). However this kit does include a detector antibody and standard protein for use with dual GST 6XHis-tagged proteins."

What this means is that if you want to used the kit to detect a protein that is only His tagged, then you also need to use a separate rabbit polyclonal antibody against His aswell (this antibody is NOT supplied with the kit).

I hope that makes sense.

a suitable rabbit polyclonal antibody that can be used in conjunction with the kit would be ab9108:

https://www.abcam.com/6X-His-tag-antibody-ChIP-Grade-ab9108.html



If there is anything else I can help you with then please let me know.

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Answer

1) How is the His tag antibody coated to the plate?
Direct coating method

2) Are there any references for the standard provided in the kit?
We have no literature references at this time.

3) Can the standard in the kit be sold separately?
Unfortunately we cannot sell the standard as a separate component. Information in regards to the standards and proteins used in this kit are considered confidential

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Answer

You are correct in your understanding of this product. The detector antibody is against GST and your sample will need a GST tag in order to be recognized by this kit. If you were to use another antibody for detection you would want this to be from a rabbit host as the HRP label is against rabbit IgG. I do not think that use of a second His tag would be appropriate with this product as these tags are would be recognized by the capture antibody and would not be presented for the detection antibody. If antibodies to your protein of interest are unavailable we can recommend custom antibody creation by our Epitomics group. More information about these services may be found at: http://www.epitomics.com/services/ or by calling 1-877-772-2622 ext.803

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Answer

Thank you for your enquiry.

We do not sell this component separately. However, I can suggest you could consider using ab89494 as standard protein for use with ab128573 6XHis-tag ELISA Kit. I can recommend reviewing the online datasheet for further information:

ab89494 GST protein (His tag)
https://www.abcam.com/index.html?datasheet=89494 (or use the following: https://www.abcam.com/index.html?datasheet=89494).

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

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Question
Answer

We tested compatibility of the kit with 10% Normal Goat serum. Recovery is 75%.

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Answer

I understand your confusion. Some of the language of the protocol is a little difficult to understand.

The ELISA kti contains two antibodies: one that is coated onto the wells which is anti-His tag. The detector antibody in the kit is rabbit anti-GST, and this is in turn detected by an anti-rabbit IgG antibody conjugated to HRP. Addition of HRP substrate produces the colored reaction.

I think this is the part of the ELISA description that is confusing:

"Bound proteins can be detected using rabbit antibody specific to the 6XHistagged fusion protein of choice (not included)."

This is confusing, since the kit obviously will not include an antibody against a unique protein. The note is meant to suggest to users that they could use the anti-IgG secondary antibody that is included with the kit, conjugated to HRP, to detect their own rabbit antibody raised against their protein of interest, if they had such an antibody.

This is repeated in the next part of the introduction:

"Standards and samples are pipetted into the wells and the 6XHis-tag present in the sample is bound to the wells by the immobilized antibody. The wells are washed and a rabbit analyte specific detector antibody is added. After washing away unbound detector antibody, an HRP-conjugated antibody specific for the rabbit antibody is pipetted to the wells."

An analyte-specific detector antibody is not in the kit. What the kit does contain is stated in the earlier sentence:

"However, this kit does include a detector antibody and standard protein for use with dual GST 6XHis-tagged proteins." The detector antibody is anti-GST.

I hope this helps. Please let me know if you have more questions.

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Answer

Thank you for contacting us.

I have searched our catalog and found the following HMGB1 antibody that binds to the desired region of interest: ab104774.
https://www.abcam.com/HMGB1-antibody-ab104774.html

It was raised against a synthetic peptide, corresponding to a region within the internal sequence amino acids 100-150 of Human HMGB1. The box B is locatedbetween residues 95-163.

Since this antibody has not yet been tested in ELISA, you are eligible for our testing discount program:
For UNTESTED species and/or applications, we have established a testing discount program. Here is a brief description of how it works:

The testing discount program is for customers who like to use an antibody/protein/kit on an untested species/application. You would purchase the antibody/protein/kit at full price, test it and submit an Abreview with your data (positive or negative). On your next order you will receive a discount for ONE antibody/protein at the full price (100%) of the antibody/protein you have tested, or a full price discount for the amount paid for the kit. The terms and conditions applicable to this offer can be found here: https://www.abcam.com/collaborationdiscount.
This programapplies to ELISA kits and other kits we offer.

Please let me know if I shall send you a discount code.


I also checked for other His-tag ELISA kit, but unfortunately we carry only ab128573.

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

Use our products? Submit an Abreview. Earn rewards!
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Answer

Thank you for contacting us.

Yes, the ELISA kit will detect His-tagged proteins.

Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you very much for your inquiry.

I have to confirm that the Anti 6XHis-tag Microplate (8 x 12 antibody coated strips) from ab128573 is not sold separately.

I am sorry I did not have a positive answer for you on this occasion and wish you a good weekend.

Read More

Answer

Thank you for your reply.

The lab has sent me the following information and advice:

This kit can be used to measure recombinant/expressed proteins tagged with 6xHis. Two examples are shown in the datasheet: GSK3B-his tagged and GST-his tagged.



The kit does work on serum (see image at the bottom of datasheet with 75% recovery rate). However, since the serum is from mouse samples, there will be too much background. What we have seen in the literature, is that researchers use these type of kits with serum samples to detect humoral response against His-tag. In this case, the researcher adds the serum as a detector antibody and not as a sample. In this case the sample would be the recombinant protein provided. But if they want to use the serum as a sample it won’t work due to mouse on mouse issues.



One potential idea could beto first deplete the serum sample from IgG molecules by incubating for a couple of hours on a GAM coated plate. Then it could be transferred to the His Tag plate. Although, it might be difficult to remove all IgG completely, it would most likely be only a depletion. However, if combined with a WB, this might work.

Another method would beNickel coated plates (which we unfortunately don't carry). This will probably work a lot better for you (just one step without having to deplete the sample of IgG).

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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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1-10 of 11 Abreviews or Q&A

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