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NEK7 GST Tag Protein, Human

NEK7 GST Tag Protein, Human

Catalog Number: UA085018 Brand: UA BIOSCIENCE
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Product Details

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.
2. He, Y., Zeng, M.Y., Yang, D., Motro, B., & Núñez, G. (2016). NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux. Nature, 540(7631), 123-127.
3. Kim, S., Lee, K., & Rhee, K. (2007). NEK7 is a centrosomal kinase critical for microtubule nucleation. Biochemical and Biophysical Research Communications, 360(1), 56-62.

Background

NEK7 (NIMA-related kinase 7) is a serine/threonine kinase belonging to the NEK family. It plays a crucial and specific role in regulating cell cycle progression, particularly in mitotic entry and spindle assembly. Structurally distinct from other NEK kinases, NEK7 contains an N-terminal catalytic kinase domain and a C-terminal regulatory domain. Its activity is tightly controlled by cell cycle-dependent phosphorylation and its interaction with NEK9. The NEK9/NEK6/NEK7 cascade is a key pathway for mitotic centrosome separation and spindle formation.

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

  1. Kinase assay buffer(5X): 200 mM Tris-HCl, pH 7.4, 100 mM MgCl2 and 0.5 mg/mL BSA, 250 μM DTT

  2. Kinase assay buffer(1X): 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 0.1 mg/mL BSA, 50 μM DTT

  3. NEK7 GST Tag Protein, Human

  4. ADP-Glo Kinase Assay (Promega, Catalog # V6930)

  5. Substrate: Casein substrate (Sinobiological, Catalog # C03-54N)

  6. Solid white multi-well plate (384-well plate) (Corning, Catalog #3572)

  7. Plate reader (PerkinElmer)

Procedure

  1. 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

  1. 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.

  2. 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.

  3. Add 5 µL ADP-Glo Reagent to the completed reaction, mix briefly, and incubate at room temperature (22–25℃) for 40 minutes.

  4. Add 10 µL Detection Reagent, and incubate the plate at room temperature (22–25℃) for 30 minutes.

  5. Read the chemiluminescent signal using the endpoint mode.

  6. Calculate the specific activity.

Standard Curve

  1. Dilute ATP and ADP to 25 μM in Kinase Assay Buffer (1×).

  2. 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

  1. Add 5 μL ADP-Glo Reagent to the completed reaction wells, mix briefly, and incubate at room temperature (22-25°C) for 40 minutes.
  2. Add 10 μL Detection Reagent, and incubate at room temperature (22-25°C) for 30 minutes.
  3. Read the chemiluminescent signal in endpoint mode.
  4. Detect the luminescent signal and generate a standard curve.

Specific Activity (pmol/min/μg) =

ATP (pmol)-Blank

Incubation time(min) ×amount of enzyme (μg)


Picture

Bioactivity

NEK7 GST Tag Protein, Human: The specific activity of NEK7 was determined to be >6.0 nmol/min/mg as per activity assay protocol.

SDS-PAGE

2μg (R: reducing condition, N: non-reducing condition).

SEC-HPLC

The purity of NEK7 GST Tag Protein, Human is more than 95% determined by SEC-HPLC.