Jun 19, 2020
Kinase-activity tagged (KAT) western blotting
- Masumi Eto1,
- Shuichi Katsuki1,
- Yoshinori Tanaka1,
- Kosuke Takeya1
- 1Okayama University of Science
Protocol Citation: Masumi Eto, Shuichi Katsuki, Yoshinori Tanaka, Kosuke Takeya 2020. Kinase-activity tagged (KAT) western blotting. protocols.io https://dx.doi.org/10.17504/protocols.io.bhpzj5p6
Manuscript citation:
Biotechniques, 2020 Apr;68(4):211-213.
doi: 10.2144/btn-2019-0136.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 19, 2020
Last Modified: June 19, 2020
Protocol Integer ID: 38361
Materials
- 10 %-formaldehyde neutral buffer: Nacalai Tesque Inc. #37152-51
- Anti-phospho (P)-Tyr antibody: Clone PY20, Zymed Laboratories Inc
- Anti-phospho (P)-PHI-1 (Thr57) antibody: Affinity-purified custom made antibody, Aves Labs Inc
- HRP-labeled anti-chicken IgY: Jackson ImmunoResearch Laboratories, Inc
- HRP-labeled anti-mouse IgG: Jackson ImmunoResearch Laboratories, Inc
- ECL solution: Pierce Supersignal WestPico plus®.
- Coomassie Brilliant Blue solution: Takara CBB Protein Safe Stain®
- Equipment used: Chemiluminescence imager: GE Amersham Imager 680
- Abbreviation: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; Gu, guanidine; PTM, posttranscriptional modification; TCEP, tris(2-carboxyethyl)phosphine); HRP, horse radish peroxidase
- Buffered 2-propanol: 20 % 2-propanol, 50 mM Tris-HCl, pH 8.0
- 6M Gu-HCl buffer: 6 M guanidine HCl, 10 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 0.5 mM TCEP
- 3M Gu-HCl buffer: 3 M guanidine HCl, 10 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 0.5 mM TCEP
- 0.1M Gu-HCl buffer: 0.1 M guanidine HCl, 10 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 0.5 mM TCEP
- Renaturation buffer: 10 mM MgCl2, 0.05 % Tween20, 50 mM Tris-HCl, pH 8.0, 0.5 mM TCEP
- Phosphorylation buffer: 10 mM MgCl2, 1 mM EGTA, 20 mM MOPS-NaOH, pH 7.0, 0.5 mM TCEP
- 0.1M ATP, pH 7.0
- Washing solution: PBS plus 0.02 % Tween20
- Blocking solution: PBS plus 1.0 % bovine serum albumin and 0.1 % Tween20
- Antibody dilution solution: PBS plus 0.1 % bovine serum albumin and 0.1 % Tween20
PROCEDURE
PROCEDURE
Experiment is conducted at room temperature unless noted.
Preparation of Laemmli Gel and Laemmli samples using a conventional method.
Running the SDS-PAGE with the samples (10-50µg of total lysates) and colored Mw marker and run electrophoresis.
Preparation of the blotting using PVDF membrane by a standard western blotting protocol.
Washing the membrane for 5 min with 10mL of Buffered 2-propanol.
Denaturation for 15min with 10 ml of 6 M Gu-HCl buffer.
Serial incubation each for 10 min with 10 mL of 3 M Gu-HCl buffer and then 10 mL of 0.1 M Gu-HCl buffer.
Washing for 3x 5min with 10 ml of Renaturation buffer.
Place the membrane in Hybri-bag
Incubation overnight with 2 ml of 0.2mg/mL substrate protein in Renaturation buffer at 4°C.
Rinse with 10 ml of Phosphorylation Buffer.
Incubation for 1h with 2 mL of Phosphorylation Buffer including 1mM ATP using 37°C shaker.
Rinse with PBS.
Incubation for 20 min with 10 mL of 10 % buffered formalin.
Rinse with PBS.
Quenching for 10 min with 10 mL of 0.1 M glycine in PBS.
Blocking for 30 min with Blocking solution.
Washing for 5 min x4 with 20 mL of Washing solution.
Incubation for overnight at 4 °C with primary antibody diluted with Antibody dilution solution.
Washing for 5 min x4 with 20 mL of Washing solution.
Incubation for 45 min with secondary antibody conjugated with HRP (diluted at 1:5,000 with Antibody dilution solution)
Washing for 5 min x4 with 20 mL of Washing solution.
Incubation for 5 min with 2 mL of ECL solution.
Imaging using chemiluminescence imager
Rinse the blot with distilled H2O twice
Staining the blot with Coomassie Brilliant Blue solution or others.
Troubleshooting
- For the first assay, a negative control blot, such as no ATP treatment, is highly recommended.
- Synthetic peptides conjugated with a carrier protein may be used as substrates.
- Renaturation efficiency may vary among kinases.