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

Play Video

Citar este artigo
Quantitative Dot Blot to Estimate Target Proteins in a Tissue Lysate. J. Vis. Exp. (Pending Publication), e21366, doi: (2023).

View Video