Difference between revisions of "Part:BBa K3113008"
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<h2>Characterization</h2>
 
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<h3>Standard Curve</h3>
  
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The resulting linear equation of the calibration curve can be used to calculate the “fmol HiBiT equivalents” in other wells with samples of interest. While one fmol HiBiT control protein does not necessarily stoichiometrically correspond to one fmol of our HiBiT-tagged proteins, their absolute amounts have a linear relationship proportional to their respective luminescence signals and thus allow us to compare total expression and secretion levels of different vesicle constructs. These calibration curves are therefore used to calculate the total amount of exported HiBiT-signal.
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<img src="https://2019.igem.org/wiki/images/d/da/T--Munich--HiBiT_calibration_curve.png" width="50%" class="figure-img img-fluid rounded" alt=" " style="margin: 2em 0;">
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<h3>Effect of Triton-X-100 on HiBiT</h3>
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Our vesicles are lysed with detergent and heat treatment to make the HiBiT tag accessible for detection. As the detergent used (Triton X-100) can interfere with luciferase activity, we tested its effect on the HiBiT assay readout to ensure we do not create measurement artefacts. For this, we mixed 60 pmol of free HiBiT peptide with increasing amounts of Triton X-100 and measured the luminescence. As can be seen in Figure S1, up to 0.1% Triton X-100 are well tolerated, whereas higher amounts of detergent have an inhibitory effect on the assay. Therefore, all samples analyzed using the HiBiT system were diluted to a final concentration of 0.05 % Triton X-100 before performing the assay to avoid introducing measurement artefacts.
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<img src="https://2019.igem.org/wiki/images/5/5a/T--Munich--Triton_X-100_Effekt.png" width="50%" class="figure-img img-fluid rounded" alt=" ">
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Revision as of 00:15, 22 October 2019


HiBiT

HiBiT is a peptide tag used for antibody-free endogenous protein detection. It is 11 amino acids long and binds with high affinity to another larger subunit called LgBiT. The bound complex has luciferase activity. Promega has all rights to this sequence.

Usage

HiBiT allows you to quantify the amount of tagged protein relatively quick via a luminescence assay. We are using this part fused to the vesicle forming proteins to measure the export rate of vesicles and the efficiency of purification.

Biology

“The Nano-Glo® HiBiT Lytic Detection System quantifies cellular protein in minutes with high sensitivity and a broad dynamic range using a simple add-mix-read assay format.”[1] The HiBiT peptide is an 11 amino acid long tag that can be added to the protein of interest. The tagged protein can be measured with the Nano-Glo® HiBiT Detection Reagent which contains the LbBiT. Together HiBiT and LgBiT form a functioning luminescent NanoBiT® enzyme.

Characterization

Standard Curve

The resulting linear equation of the calibration curve can be used to calculate the “fmol HiBiT equivalents” in other wells with samples of interest. While one fmol HiBiT control protein does not necessarily stoichiometrically correspond to one fmol of our HiBiT-tagged proteins, their absolute amounts have a linear relationship proportional to their respective luminescence signals and thus allow us to compare total expression and secretion levels of different vesicle constructs. These calibration curves are therefore used to calculate the total amount of exported HiBiT-signal.

Effect of Triton-X-100 on HiBiT

Our vesicles are lysed with detergent and heat treatment to make the HiBiT tag accessible for detection. As the detergent used (Triton X-100) can interfere with luciferase activity, we tested its effect on the HiBiT assay readout to ensure we do not create measurement artefacts. For this, we mixed 60 pmol of free HiBiT peptide with increasing amounts of Triton X-100 and measured the luminescence. As can be seen in Figure S1, up to 0.1% Triton X-100 are well tolerated, whereas higher amounts of detergent have an inhibitory effect on the assay. Therefore, all samples analyzed using the HiBiT system were diluted to a final concentration of 0.05 % Triton X-100 before performing the assay to avoid introducing measurement artefacts.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

References

  1. ↑ https://www.promega.de/products/protein-quantitation-and-detection/protein-quantitation/nano-glo-hibit-lytic-detection-system/?catNum=N3030