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Description
NEK7 GST Tag Protein, HumanProduct Specification Species Human Synonyms NimA related protein kinase 7, Never in mitosis A related kinase 7 Accession Q8TDX7 Amino Acid Sequence Met1 Ser302 with GST Tag at the N Terminus Expression System E. coli Molecular Weight 55 70kDa (Reducing) Purity 90% by SDS PAGE & > 90% by HPLC Conjugation Unconjugated Tag GST Tag Physical Appearance Liquid Storage Buffer 50mM Tris, 150mM NaCl, pH7. 5, 1mM DTT, 10%Glycerol Stability & Storage Stable for
Product Specification
| Species | Human |
| Synonyms | NimA-related protein kinase 7, Never in mitosis A-related kinase 7 |
| Accession | Q8TDX7 |
| Amino Acid Sequence | Met1-Ser302 with GST Tag at the N-Terminus |
| Expression System | E.coli |
| Molecular Weight | 55-70kDa (Reducing) |
| Purity | ＞90% by SDS-PAGE & > 90% by HPLC |
| Conjugation | Unconjugated |
| Tag | GST Tag |
| Physical Appearance | Liquid |
| Storage Buffer | 50mM Tris, 150mM NaCl, pH7.5, 1mM DTT, 10%Glycerol |
| Stability & Storage | Stable for 12 months upon stored at -80℃ from the date of receipt. And avoid repeated freeze-thaws cycles. |
| Reference | 1. Shi, H., et al. (2016). NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7. Nature, 540(7631), 440-445. |
Background
Protocol
Experimental Method
Assay Principle: The ADP-Glo™ Kinase Assay Kit is used to measure NEK7 activity. This kit quantitatively detects the amount of ADP produced by the NEK7 reaction. The specific steps are as follows: First, add ADP-Glo™ Reagent to terminate the kinase reaction and deplete the remaining ATP; then add the Kinase Detection Reagent to convert ADP back to ATP, and detect the newly synthesized ATP through a luciferase/luciferin reaction system.
Materials
Kinase assay buffer(5X): 200 mM Tris-HCl, pH 7.4, 100 mM MgCl2 and 0.5 mg/mL BSA, 250 μM DTT
Kinase assay buffer(1X): 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 0.1 mg/mL BSA, 50 μM DTT
NEK7 GST Tag Protein, Human
ADP-Glo Kinase Assay (Promega, Catalog # V6930)
Substrate: Casein substrate (Sinobiological, Catalog # C03-54N)
Solid white multi-well plate (384-well plate) (Corning, Catalog #3572)
Plate reader (PerkinElmer)
Procedure
Prepare the Substrate/ATP mixture as follows (using 25 μM as an example):
Sample Name |
Amount (μL) |
10 mM ATP Solution |
1 |
Kinase Assay Buffer III (5x) |
79 |
Substrate at 1 mg/mL |
80 |
Dilute NEK7 to 15 µg/mL, 7.5 µg/mL, and 3.75 µg/mL using Kinase Assay Buffer (1x), and add 3 µL to each well of a 384 -well plate.
Initiate the reaction by adding 2 µL of the assay mixture prepared in Step 1 to each well. Set up a blank control containing only 3 µL of Kinase Assay Buffer (1x). The total reaction volume is 5 µL. Incubate the reaction at room temperature (22–25℃) for 40 minutes.
Add 5 µL ADP-Glo Reagent to the completed reaction, mix briefly, and incubate at room temperature (22–25℃) for 40 minutes.
Add 10 µL Detection Reagent, and incubate the plate at room temperature (22–25℃) for 30 minutes.
Read the chemiluminescent signal using the endpoint mode.
Calculate the specific activity.
Standard Curve
Dilute ATP and ADP to 25 μM in Kinase Assay Buffer (1×).
Mix 25 μM ATP and 25 μM ADP as shown in the table below to prepare the ATP+ADP mixture, and add 5 μL to each well of a 384-well plate.
Well Number |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
25μM ADP (μL) |
100 |
80 |
60 |
40 |
20 |
10 |
5 |
4 |
3 |
2 |
1 |
0 |
25μM ATP (μL) |
0 |
20 |
40 |
60 |
80 |
90 |
95 |
96 |
97 |
98 |
99 |
100 |
- Add 5 μL ADP-Glo Reagent to the completed reaction wells, mix briefly, and incubate at room temperature (22-25°C) for 40 minutes.
- Add 10 μL Detection Reagent, and incubate at room temperature (22-25°C) for 30 minutes.
- Read the chemiluminescent signal in endpoint mode.
- Detect the luminescent signal and generate a standard curve.
Specific Activity (pmol/min/μg) = |
ATP (pmol)-Blank |
Incubation time(min) ×amount of enzyme (μg) |
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