High affinity NanoBiT (HiBiT) -Is now available. Thank you to all evaluators!

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  • Last Post 22 August 2017
alandrem posted this 12 April 2016

To learn more visit: www.Promega.com/HiBiT

In contrast to the low affinity interaction between LgBiT and SmBiT used to study protein interactions, the NanoBiT system was also developed with a small peptide (HiBiT) that has high affinity for the LgBiT subunit. The 11 aa HiBiT peptide interacts spontaneously with LgBiT to give a bright luminescent enzyme. Promega is currently developing products based on this high affinity interaction including 1) lytic detection reagents for the HiBiT tag; 2) live cell reagents to detect HiBiT on surface-expressed or secreted proteins; 3) HiBiT blotting reagents that enable blots to be performed in just a few minutes; and 4) live cell detection of the intracellular HiBiT:LgBiT interaction.

Would you like to learn more or become and early evaluator? We currently have test materials available. Please leave a comment to indicate your interest and we’ll get back to you.

 

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acashikar posted this 10 June 2016

What is the difference in binding constants between high and low affinity NanBiTs?

alandrem posted this 10 June 2016

The low affinity interaction has a Kd of 190uM, the high affinity interaction has a Kd of 700pM

Allisonchi posted this 19 July 2016

Hi. We are interested in evaluating HiBit system if it's still available.

alandrem posted this 25 July 2016

Great! Yes, we still have HiBiT materials available for evaluation. I will reach out to you with some additional information so that we get the correct materials sent out to you for your research application. Thanks for your interest. 

SheridanC posted this 20 October 2016

Hello, I am very interested in trying out the HiBiT technology. Is it still available?

Thanks,

alandrem posted this 20 October 2016

Hi SheridanC, Thanks for interest in HiBiT. Yes, we are still looking for researchers interested in trying this technology. I will reach out to you to find out more about your application so that we can send you the correct materials.

mokros posted this 30 November 2016

Will the lytic detection reagents be also available for the standard NanoBiT assay? I would be quite keen on trying that.

BBinkowski at Promega posted this 30 November 2016

"Will the lytic detection reagents be also available for the standard NanoBiT assay? I would be quite keen on trying that."

Mokros: The NanoBiT assay for protein:protein interactions uses a live cell, non-lytic assay protocol. This allows protein interactions to be studied in their native cellular context in real time. A lytic detection reagent can promote the dissociation of a protein:protein interaction (dilution of cellular contents, presence of detergents, etc.). Promega doesn't recommend use of a lytic detection reagent for these reasons.

pbrescia posted this 26 January 2017

Hello, I am very interested in measuring the stability of a protein dimer formation. Would it be feasible using the HiBiT technology?

Thanks.

alandrem posted this 26 January 2017

Hi @pbrescia,

I think for protein dimer formation the low affinity NanoBiT system might be better. One member of the dimer would be tagged with SmBiT and the other with LgBiT. If they are in dimer formation you would have the active NanoBiT enzyme, but if the  protein dimer fell apart you would lose the enzyme and the signal. There are some example of this in this poster: NanoBiT poster

Most of the dimers studies we have done only go for 1-2hrs. However, we are working on some new substrates that should allow for longer studies 12-24hrs if you wanted to monitor the status of the dimer for longer periods of time.

Does that help?

 

 

 

JALopez posted this 01 March 2017

Hi, today at my department seminar (UofIowa) I saw the HiBiT technology being used for detecting surface-expressed proteins in HEK cells. I'm really interested to know more about this application and looking into developing something similar in my lab. Let me know if it is still possible to evaluate the HiBiT system. 

Thank you 

alandrem posted this 06 March 2017

Hi @JALopez. Great to hear you learned about HiBiT technology at your seminar. Detection of surface-expressed proteins is an application that should be well suited to the use of HiBiT. Yes, we do still have evaluation material available. Typically we provide the cloning vectors and also some detection reagent for you to give it a try. I will reach out via email to confirm your interest and get your shipping details. Thanks for your interest!

ppfusion posted this 14 March 2017

Hi, we would be interested in evaluating the high affinity HiBiT:LgBiT system for forcing protein interactions within cells, if this is still available. Thanks!

alandrem posted this 15 March 2017

Hello @ppfusion. Thanks for your interest in the HiBiT system. We do still have materials available. If I understand correctly you would like to tag one protein with LgBiT and one with HiBiT and express them in the same cell. The LgBiT:HiBiT interaction would force together your tagged proteins. This isn't something that we have tried but sounds interesting. We do still have materials available. I will follow up with you to get your shipping details etc.

aliceyin902 posted this 20 April 2017

I am very interested in using it for protein: protein interaction. Particularly monitor the interaction over a time course. I would like to evaluate it with my proteins if this is still available! Thank you very much!

alandrem posted this 21 April 2017

Hello @aliceyin902. Thank you for joining the NanoBiT forum and for your interest in using NanoBiT for your protein interaction studies!  The material that we currently have for evaluation is our HiBiT tagging system. This utilizes the high affinity interaction between the HiBiT tag and LgBiT and can basically be used as a quantitative protein tagging method. Your protein is tagged with HiBiT and detection reagents contain purified LgBiT and substrate  give a luminescent signal.  For protein interactions we recommend using the low affinity interaction between SmBiT and LgBiT, where the interaction of the two subunits is facilitated by the proteins you are studying. Constructs to make SmBiT and LgBiT fusions to your proteins of interest are available in our catalog, N2014 and N2015. Hope this helps but please let me know if you have additional questions.

cornelisdehaan posted this 17 June 2017

Hi, I am interested in using the HiBit technology to study transport of viral proteins to the cell surface and subsequent assemble into virus(-like) particles. Hopefully, you are still looking for evaluators.

alandrem posted this 20 June 2017

Hello @cornelisdehann,

Thanks for joining the forum. We are getting close to the initial HiBiT product launch, however we do still have materials available! Your application sounds interesting. HiBiT seems well-suited to virology field and it will be interesting to see the different things it can enable.  I can follow-up with you get shipping information etc so that we can get some materials out to you. Thanks!

zwynorthern posted this 12 July 2017

Hi, not sure if it is too late to request materials for evaluation. I am interested in using this system to evaluate the process of virus entry and fusion. Please let me know if the sample is still available. Thank you!

alandrem posted this 14 July 2017

Hello @zwynorthern,

Thanks for joining the forum and for expression your interest in the HiBiT system. Seems to be of high interest among the viral research community. Yes, we do still  have materials that can be provide for your viral entry and fusion studies. I can follow up with you to get the ball rolling on getting materials out to you. Thanks!

JackChu posted this 16 July 2017

Hi, not sure if it is too late to request materials for evaluation. I am interested in using this system to see if we could tag and trace endogenous dynamic protein expression and/or localization. I am curious if the 11aa HiBiT tag would influence endogenous protein half-life? If it is still available, please let me know. Thanks!

alandrem posted this 17 July 2017

Hello @JackChu,

Thanks for your post. We have been having good success knocking-in the HiBiT tag using CRISPR/CAS9 for endogenous protein tagging. It should work well for studying the dynamics of protein expression. It may not work that well for studying subcellular localization because the bioluminescence is still difficult to image with standard microscopy. I will follow up with you to confirm your interest and get materials out to you if appropriate. Thanks!

 

alandrem posted this 17 July 2017

Sorry, I forgot to address your question about protein half-life. Thus far we have not seen any noticeable impact on protein biology in the systems that we have tested. However, I do not know if we have performed traditional protein 1/2-life studies. I can look into this.

ouyangy posted this 18 August 2017

Hi, I'm very interested in testing LgBiT and HiBiT interaction to measure protein entry into cells. Is that possible to get an aliquot of the plasmid and some detailed information from you? you can contact me by my registered email. thank you very much!

alandrem posted this 22 August 2017

Hello @ouyangy. Thanks for posting to the forum. Yes, I will contact you to learn a little more about your intended experiment.

 

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