Quantitative Dot Blot to Estimate Target Proteins in a Tissue Lysate

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

1. 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).
2. Homogenize the tissue with a homogenizer for 1 min on ice.
3. Centrifuge for 10 min at 8,000 x g at 4 °C.
4. Collect the supernatant into new tubes (1.5 mL) and take out 1 µL for pro....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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