The wells of a 96-well microtiter plate (Immulon 2, Dynatech Laboratories, Inc.) were coated overnight at 4 °C with 0.1 ml of 1 mg/ml rat tail collagen I (Corning) or mouse collagen IV (Corning) in 0.09% acetic acid. The wells were washed twice with 0.15 ml TBS and then blocked for 1 h at room temperature with 0.15 ml of 100 mg/ml bovine serum albumin (Sigma) in TBS. Recombinant GST-tagged I domains were serial diluted from 1 mM in various wash buffers (TBS containing 0.05% Tween-20, 10 mg/ml BSA, and either 5 mM EDTA, 1 mM CaCl2, or 1 mM MgCl2). The wells were washed once with 0.15 ml of the appropriate wash buffer, and then 0.1 ml of each I domain dilution was added and allowed to interact for 1.5 h at RT. Wells were washed three times with 0.15 ml of the appropriate wash buffer. Then 0.1 ml of a 1:1000 dilution of anti-GST HRP conjugate (GE Healthcare) in the appropriate wash buffer was added for 1 h at RT. Following incubation, the wells were washed three times, and then 0.06 ml of 3, 3′,5,5′-tetramethylbenzidine substrate (Sigma) was added for 1 h at RT. Reactions were stopped with 0.015 ml of 4 N H2SO4, and the plates were read at 450 nm. Representative binding plots are included in supplemental information (Fig. S8 ).
Anti gst hrp conjugate
Manufactured by GE Healthcare
The Anti-GST HRP conjugate is a laboratory reagent used to detect and quantify proteins fused with Glutathione S-Transferase (GST) in various applications. It contains an antibody against GST that is conjugated with Horseradish Peroxidase (HRP), enabling colorimetric or chemiluminescent detection of GST-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Rat tail collagen 1, by Corning (1 mentions)
Mouse collagen 4, by Corning (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
3 3 5 5 tetramethylbenzidine substrate, by Merck Group (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
1 step ultra tmb elisa substrate solution, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
Ezwestblue, by ATTO Corporation (1 mentions)
2d silver stain reagent 2, by Cosmo Bio (1 mentions)
4 protocols using anti gst hrp conjugate
1
Characterization of I Domain Binding
2
Measuring Fc Variant Binding to FcγRs
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Example 12
In this example, the binding forces of the Fc variants to FcγRs were measured. Specifically, 50 μl of each of the IgG Fc variants diluted to 4 μg/ml with 0.05 M Na2CO3 (pH 9.6) was immobilized onto a flat-bottom polystyrene high-bind 96-well microplate (costar) at 4° C. for 16 h, blocked with 100 μl of 4% skim milk (GenomicBase) (in 0.05% PBST pH 7.4) at room temperature for 2 h, and washed four times with 180 μl of 0.05% PBST (pH 7.4). Thereafter, 50 μl of FcγRs serially diluted with 1% skim milk (in 0.05% PBST pH 7.4) was plated in each well and the reaction was carried out at room temperature for 1 h. After washing, an antibody reaction with 50 μl of anti-GST-HRP conjugate (GE Healthcare) was allowed to proceed at room temperature for 1 h. The plate was washed and developed with 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific). The reaction was quenched with 2 M H2SO4 (50 μl each). Then, the reaction product was analyzed using an epoch microplate spectrophotometer (BioTek). Each experiment was conducted in duplicate.
3
In vitro Ubiquitylation of HEC2 by COP1
4
Recombinant Protein Detection by Western Blot
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For detection of recombinant proteins, the elution fractions were processed by SDS-PAGE and blotted onto PVDF membranes (FluoroTrans; Nihon Pall Ltd., Tokyo, Japan). Nonspecific binding of the antibodies was blocked with EZBlock reagent (ATTO Corp., Tokyo, Japan) overnight at 4 °C. The blots were washed three times with washing buffer (0.15 M PBS, pH 7.3, 0.1% Tween 20). The recombinant His-CSA proteins on the PVDF membranes were cross-reacted with a rabbit anti-spinach OASTL antibody [26] for 1 h. After washing three times for 10 min each, the blots were incubated for 60 min with a goat anti-rabbit IgG-HRP (Tanpaku Seisei Kogyo, Isezaki, Japan) at a 1:2000 dilution. The blots were washed as before and the colour was developed by adding EzWestBlue (ATTO Corp.). For detection of GST proteins, the blots were incubated with Anti-GST-HRP Conjugate (GE Healthcare) and the colour was developed as described above. When GST-CSLP proteins were detected, the gels were stained with silver nitrate using the 2D-Silver Stain Reagent II (Cosmobio, Tokyo, Japan).
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