A2AR samples were denatured with 5x Laemmli buffer and incubated for 20 min at RT prior to analysis without heating to avoid aggregates formation. Proteins were separated by SDS-PAGE on a 4–15% acrylamide gel (4–15% Mini-PROTEAN® TGX Stain-Free™ Gel, Bio-Rad) and subsequently immobilized by electro-transfer to PVDF membrane. The immunodetection of A2AR was performed by using the SNAP i.d. system (Millipore) with either a primary A2AR antibody (mAb 7F6-G5-A2), Santa Cruz Biotechnology) or an anti-His HRP antibody. Quantification of the signal was performed using Image Lab 4.1 software from Bio-Rad to evaluate the extraction efficiency. SDS-PAGE were silver stained using Bio-Rad Dodeca Silver Stain Kit following supplier protocol or coomassie stained using the PageBlue™ protein staining solution.
Dodeca silver stain kit
Manufactured by Bio-Rad
Sourced in United States
The Dodeca Silver Stain Kit is a laboratory tool designed for the detection and visualization of proteins in polyacrylamide gels. The kit provides a sensitive and reliable method for staining protein bands, enabling researchers to analyze and quantify their samples with accuracy.
Automatically generated - may contain errors
Lab products found in correlation
Snap i d system, by Merck Group (1 mentions)
Mini protean tgx stain free gel, by Bio-Rad (1 mentions)
Image lab 4, by Bio-Rad (1 mentions)
Immobilon western, by Merck Group (1 mentions)
Las4000 luminescence imager, by Fujifilm (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
Ni sepharose 6 fast flow, by GE Healthcare (1 mentions)
5n naoh, by Merck Group (1 mentions)
Mini protean 3 cell, by Bio-Rad (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
Image scanner, by Cytiva (1 mentions)
5 protocols using dodeca silver stain kit
1
Adenosine A2A Receptor Immunodetection
2
Characterizing Oxidized DJ-1 Protein
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Protein
samples were denatured in the SDS sample buffer [63 mM Tris-HCl (pH
6.8) containing 0.012% bromophenol blue, 5% sucrose, and 2% SDS] for
5 min at 95 °C (nonreduced condition). To reduce protein samples,
1% beta-mercaptoethanol (2-ME) was added to SDS sample buffer and
then heat treatment was conducted. After separation of the samples
by SDS-PAGE (12.5% gel), the proteins were transferred to an Immobilon-P
Transfer Membrane (Millipore, Bedford, MA) for the western blot analysis.
The membranes were blocked in a Tris-buffered saline (pH 7.4) containing
0.1% Tween 20 (TBS-T) containing 5% skimmed milk powder (Snow Brand
Milk Products, Tokyo, Japan), incubated with anti-oxDJ-1 mAb (clone
M106, 1 μg/mL, ref (18 (link))), mouse anti-DJ-1 mAb (clone 3E8, 1 μg/mL, Medical
& Biological Laboratories, Nagoya, Japan), or anti-β-actin
mAb (AC-15) at 4 °C for 18 h, and incubated with HRP-conjugated
secondary antibodies for at least 1 h. After the incubation with Immobilon
Western (Millipore), the immunoreactivity was visualized with an LAS-4000
luminescence imager (Fujifilm, Tokyo, Japan). For silver staining,
the separated proteins were stained with the Dodeca Silver Stain Kit
(Bio-Rad Laboratories, Hercules, CA).
samples were denatured in the SDS sample buffer [63 mM Tris-HCl (pH
6.8) containing 0.012% bromophenol blue, 5% sucrose, and 2% SDS] for
5 min at 95 °C (nonreduced condition). To reduce protein samples,
1% beta-mercaptoethanol (2-ME) was added to SDS sample buffer and
then heat treatment was conducted. After separation of the samples
by SDS-PAGE (12.5% gel), the proteins were transferred to an Immobilon-P
Transfer Membrane (Millipore, Bedford, MA) for the western blot analysis.
The membranes were blocked in a Tris-buffered saline (pH 7.4) containing
0.1% Tween 20 (TBS-T) containing 5% skimmed milk powder (Snow Brand
Milk Products, Tokyo, Japan), incubated with anti-oxDJ-1 mAb (clone
M106, 1 μg/mL, ref (18 (link))), mouse anti-DJ-1 mAb (clone 3E8, 1 μg/mL, Medical
& Biological Laboratories, Nagoya, Japan), or anti-β-actin
mAb (AC-15) at 4 °C for 18 h, and incubated with HRP-conjugated
secondary antibodies for at least 1 h. After the incubation with Immobilon
Western (Millipore), the immunoreactivity was visualized with an LAS-4000
luminescence imager (Fujifilm, Tokyo, Japan). For silver staining,
the separated proteins were stained with the Dodeca Silver Stain Kit
(Bio-Rad Laboratories, Hercules, CA).
3
Purification of GM2A Protein via Ni-column Chromatography
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For Ni-column chromatography, 5N NaOH (Sigma-Aldrich) was added to the CM to adjust the pH to 8.0–8.3, and then the CM was filtered with a Minisart® Syringe Filter 0.45 µm (Sartorius, Göttingen, NI, Germany). Then samples were applied to Ni Sepharose 6 Fast Flow (GE Healthcare, Little Chalfont, BKM, UK) equilibrated with 25 mM sodium phosphate buffer (NaPB), 0.3 M NaCl (pH 8.0). After washing with 25 mM NaPB, 0.3 M NaCl (pH 7.0 and 6.0), the bound proteins were eluted with 50 mM sodium acetate buffer, 0.3 M NaCl (pH 4.0). Each fraction was subjected to SDS-PAGE on a 12.5% (w/v) acrylamide gel and silver staining with a Dodeca Silver Stain Kit (Bio-Rad). The molecular weight of GM2A was calculated based on those of the APRO markers (APRO Science, Tokushima, Japan).
4
Tick Saliva Protein Profiling by SDS-PAGE and Western Blot
5
Staining Protein Gels with Silver and Coomassie
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SDS-PAGE were silver stained using Bio-Rad Dodeca Silver Stain Kit following supplier protocol or coomassie stained using the PageBlue™ protein staining solution.
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