Quantitative Dot Blot to Estimate Target Proteins in a Tissue Lysate

Overview

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 protein concentration assay with a BCA kit.
   NOTE: All the commonly used lysis buffers for western blot analysis and Dot blot analysis can be used for QDB analysis.

2. Determining the Specificity of Antibody

1. Prepare sample lysates (20 µg) in section 1.4, IgG-free BSA (20 µg), and standard protein (600 pg), for western blot analysis.
   NOTE: IgG-free BSA is used as a negative control and the standard protein is used as a positive control. The specificity of the antibody is demonstrated by showing one band of the right size using the western blot analysis.

3. Defining the Linear Range of the QDB Analysis

1. Dissolve the standard protein with ddH₂O. Dilute the protein to a concentration of 1,200 pg/µL.
2. Dilute the concentrated protein from the prepared samples in section 1.4 to 4 µg/µL.
3. Dissolve the BSA with ddH₂O. Dilute the protein to a concentration of 1,200 pg/µL and 4 µg/µL.
4. A series of dilutions of standard protein and prepared samples were prepared with the guidance of Table 1 and Table 2.
5. Mix 10 µL dilution standard protein or dilution prepared samples and 10 µL 2x loading buffer (120mM Tris-HCl pH 6.8, 20% Glycerol, 4% SDS, 0.2% Bromphenol Blue, 200 mM DTT) together.
6. Heat the mixtures at 85 °C for 5 min.

4. Process of QDB Analysis

1. Sample application
   1. Support the QDB plate to avoid the bottom of the plate touching the surface of the table. For instance, use an empty pipette tip box as support.
   2. Load up to 2 µL of the sample to the center of the membrane at the bottom of the individual unit of the QDB plate.
2. Drying the plate
   1. Leave the loaded QDB plate for 1 h at room temperate (RT) or as an alternative leave the loaded plate at 37 °C for 15 min in a well-ventilated space.
3. Blocking the plate
   1. Dip the QDB plate in the transfer buffer (0.039 M Glycine, 0.048 M Tris, 0.37% SDS, 20% methyl alcohol) and gently shake the plate for 10 s.
   2. Rinse the QDB plate gently with TBST (Tris-buffered saline, 0.1% Tween 20) three times, and then wash the plate for 5 min in TBST under constant shaking.
   3. Block the QDB plate with blocking buffer (5% nonfat milk in TBST (100 µL/well) for 1 h under constant shaking.
4. Primary antibody incubation
   1. Dilute the primary antibody in the blocking buffer at a chosen concentration (from 1: 500 to 1: 5,000), and add 100 µL to each individual well of an ordinary 96-well plate. Insert the QDB plate into the 96-well plate, and incubate the combined plates either for 2 h at RT or overnight at 4 °C under constant shaking.
   2. Alternatively, place the QDB plate inside a box, and fill the box with a blocking buffer 2 to 3 mm above the membrane portion of the plate if the same antibody is used for the whole plate. Add the primary antibody at the chosen concentration, and incubate the plate either for 2 h at RT or overnight at 4 °C under constant shaking.
   3. Rinse the plate gently with TBST three times before it is washed with TBST three times, each time for 5 min under constant shaking.
5. Secondary Antibody incubation
   1. Dilute the secondary antibody in the blocking buffer at the chosen concentration (from 1:1,000 to 1:50,000), and either aliquot 100 µL/well into a 96-well plate or in a box as described in section 4.4.2 and then incubate the QDB plate inside either the loaded 96-well plate or the box for 1 h at RT under constant shaking.
   2. Rinse the QDB plate gently 3 times with TBST, then wash the plate 3 times, 5 min each with TBST under constant shaking.
6. Quantification
   1. Prepare enhanced chemiluminescence substrate (ECL) by following the manufacturer's instructions.
   2. Aliquot ECL substrate into a 96-well plate (100 µL/well) and insert the QDB plate inside the 96-well plate for 2 min under constant shaking.
   3. Remove the QDB plate from the 96-well plate and shake briefly to remove the excess liquid. Place the plate onto a white microtiter plate.
   4. Turn on the microplate reader, and select "plate with cover" on the user interface before placing the combined plates (QDB plate + white plate adaptor) inside the microplate reader for quantification.
      NOTE: Make sure to use the white, non-transparent microtiter plate as an adaptor to avoid any interference from the plate. Make sure to choose "plate with cover" to avoid jamming the machine when combined plates (QDB plate + 96-well plate adaptor) are placed inside the microplate reader.

Table 1. Dilution Scheme for standard protein curve.

	S1 (0pg/µl)	S2 (33.3pg/µl)	S3 (100pg/µl)	S3 (300pg/µl)	S5 (600pg/µl)	S6 (1200pg/µl)
Standard protein (1200pg/µl)	0	1.39	4.17	12.5	25	50
IgG-free BSA (4pg/µl)	50	48.61	45.83	37.5	25	0
Total	50	50	50	50	50	50

Table 2. Dilution Scheme for prepared samples

	X1 (0µg/µl)	X2 (0.25µg/µl)	X3 (0.5µg/µl)	X4 (1µg/µl)	X5  (2µg/µl)	X6  (4 µg/µl)
Sample (4µg/µl)	0	3.125	6.25	12.5	25	50
IgG-free BSA (4µg/µl)	50	46.875	43.75	37.5	25	0
Total	50	50	50	50	50	50

Materials

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