Quantitative Dot Blot to Estimate Target Proteins in a Tissue Lysate
Published: May 31, 2023
Abstract
Source: Qi, X., et al. High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB). J. Vis. Exp. (2018).
This video demonstrates a quantitative dot blot, or QDB, technique for the absolute quantification of proteins of interest in a tissue sample. The proteins in the sample are immobilized on the nitrocellulose membrane inside the wells of a QDB plate. Utilizing target protein-specific antibodies labeled with a peroxidase enzyme, a chemiluminescent substrate is oxidized to produce light, which is measured to determine the protein concentration of the sample.
Protocol
1. Sample Preparation Take 50 mg mouse tissue into a 1.5 mL Tube. Add 200 µL lysis buffer (50 mM HEPES, pH 7.4, 137 mM NaCl, 5 mM EDTA, 5 mM EGTA, 1 mM MgCl2, 10 mM Na2P2O7, 1% Triton X-100, 10% glycerol), supplemented with protease and phosphatase inhibitors (100 mM NaF, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/mL pepstatin, 10 µg/mL leupeptin, 5 µg/mL aprotinin). Homogenize the tissue with a homogenizer for 1 min on ice. C…
Declarações
The authors have nothing to disclose.
Materials
| Microplate reader | Tecan | Tecan Infinite 200 PRO | |
| QDB plate | Yantai Zestern Co.Ltd | Plates are available upon request for verification purpose either through email or visiting www.zestern.net. | |
| Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H+L) (min X Bov, Ck, Gt, GP, Sy Hms, Hrs, Hu, Ms, Rat, Shp Sr Prot) | Jackson | 711-035-152 | |
| Enhanced chemiluminesence substrate | Thermo | 32134 | |
| Rabbit anti-CAPG | Sino Biologic Inc | 14213-T52 | |
| CAPG protein | Sino Biologic Inc | 14213-HNAE | |
| HEPES | Sigma | H4034 | |
| NaCl | Tianjin Ruiji Jin special Chemical Co., Ltd | 10461220 | |
| MgCl2 | Tianjin Zhiyuan Chemical Reagent Co., Ltd | 20120802 | |
| EDTA | Sigma | E9884-500G | |
| EGTA | Sigma | BNN1913 | |
| Na2P2O7 | Haituo Chinese medicine test | 20160914 | |
| Triton X-100 | Sigma | T9284 | |
| Glycerol | Sigma | G7757 | |
| Phenylmethylsulfonyl fluoride | Sigma | P7626-5G | |
| Pepstatin | Sigma | Z290033 | |
| Leupeptin | Sigma | L2884 | |
| Aprotinin | Sigma | A3428 | |
| Tris-HCl | Sigma | T6455 | |
| SDS | Sigma | L5750-500G | |
| DTT | Sigma | 43815-1G | |
| Bromphenol Blue | Sigma | B-0126 | |
| NaF | Sigma | S7920 |
Tags
Citar este artigo
Quantitative Dot Blot to Estimate Target Proteins in a Tissue Lysate. J. Vis. Exp. (Pending Publication), e21366, doi: (2023).