Antibodies were enriched from serum by passing serum over an agarose SulfoLink® affinity column (Thermo Scientific) containing covalently immobilized 1. 2 mL of resin slurry was added to a 5 mL fritted syringe and evacuated by centrifugation and washed three times in 2 mL of coupling buffer (50 mM, 5 mM EDTA, pH 8.5). 2 mg of 1 dissolved in PBS was incubated with the column for 45 minutes at room temperature. After washing in PBS containing 1 M NaCl, unreacted iodoacetamide groups were quenched with a solution of 50 mM cysteine in coupling buffer and equilibrated into TBS. Approximately 50 µL of NOD mouse serum was diluted to 500 µL in Gentle Ag/Ab Binding Buffer (Thermo Scientific) and added to the affinity column. The column was rotated for 60 min at room temperature. The column was washed three times with 2 mL TBS and bound IgG was eluted under high salt conditions using Gentle Ag/Ab Elution Buffer pH 6.6 (Thermo Scientific). The IgG was dialyzed overnight in TBS and concentrated to ≈250 µg mL−1 total protein using a 50 kDa molecular weight cut-off spin filter (EMD Millipore). SDS-PAGE gels were stained with Pierce Silver Stain Kit for Mass Spectrometry (Thermo Scientific) and imaged using a ChemiDoc™ XRS+ System (Bio Rad), which also measured band intensities to generate standard curves.
Pierce silver stain for mass spectrometry kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce Silver Stain for Mass Spectrometry kit is a laboratory product designed for the detection and visualization of proteins separated by gel electrophoresis. The kit utilizes a silver-based staining method to enhance the sensitivity of protein detection compared to traditional Coomassie blue staining. The stained proteins can then be further analyzed using mass spectrometry techniques.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Chemi lumi one super, by Nacalai Tesque (2 mentions)
Hrp conjugated secondary antibody, by Agilent Technologies (2 mentions)
Immobilon p pvdf membrane, by Merck Group (2 mentions)
Chemi lumi one ultra, by Nacalai Tesque (2 mentions)
Pierce magnetic rna protein pull down kit, by Thermo Fisher Scientific (2 mentions)
Pierce rna 3 end desthiobiotinylation kit, by Thermo Fisher Scientific (2 mentions)
Polyacrylamide gel, by Bio-Rad (2 mentions)
Triple tof 5600 system, by AB Sciex (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Gentle ag ab elution buffer ph 6, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Proq emerald glycoprotein stain, by Thermo Fisher Scientific (1 mentions)
Sypro ruby, by Thermo Fisher Scientific (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Orbitrap elite, by Thermo Fisher Scientific (1 mentions)
Mascot, by Matrix Science (1 mentions)
α ha magnetic beads, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris precast gel, by Thermo Fisher Scientific (1 mentions)
27 protocols using pierce silver stain for mass spectrometry kit
1
Affinity Purification of Serum Antibodies
2
Identifying MOAP-1 Interactors by Co-IP
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Plasmid encoding Flag-tagged MOAP-1 was transfected into 293T. Twenty-four hours posttransfection, cells were treated with or without 100 μM of etoposide for another 18 or 24 h. Cells were collected and lysed in 0.5% TritonX-100 lysis buffer (0.5% TritonX-100, 20 mM Hepes (pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, 2 mM EGTA, 2 mM DTT) supplemented with aprotinin, leupepcin, phenylmethanesulfonylfluoride and phosphatase inhibitor cocktails (Sigma) and then Flag-MOAP-1 and its interacting protein was co-immunoprecipitated by using Flag M2 agarose beads. Co-IP samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Protein was visualized with Pierce Silver Stain Kit for Mass Spectrometry (Thermo Scientific). The bands of interest were analyzed by mass spectrometry at Duke Proteomics and Metabolomics Core Facility (Durham, NC, USA).
3
Purification and Characterization of PBP2a
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Immunopurified PBP2a MRSA protein fractions were separated on 10% Tris–Glycine SDS-PAGE gels (Thermo Fisher Scientific, MA). Silver staining and destain of separated proteins was performed per manufactures instructions using the Pierce silver stain kit for mass spectrometry (Thermo Fisher Scientific, Waltham MA). In-gel peptide analysis of PBP2amecA was performed from excised gel bands. Briefly, gel bands were destained per the manufacturer’s protocol, washed in 100 mM NH4HCO3 (pH 8.0) and then acetonitrile, dried, and incubated with 50 ng of MS-grade Pierce trypsin protease or Proteinase K overnight (Thermo Fisher Scientific, Waltham MA). Peptides were extracted from gel pieces by sequential addition of NH4HCO3 (pH 8.0) and acetonitrile. Collected supernatant was dried and analyzed by bottom-up peptide analysis. Glycoprotein detection of SDS-PAGE separated proteins was performed using the Pro-Q Emerald glycoprotein stain while total protein was stained using SYPRO Ruby (Thermo Fisher Scientific, Waltham MA). Staining and visualization was performed per manufacturer’s instructions.
4
Proteomic Analysis of NDUFA4L2 Interactome
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After verifying that NDUFA4L2 was immunoprecipitated, we determined the proteins immunoprecipitated in RCC4-P versus RCC4-KO-643 cell extracts. We first loaded the immunoprecipitation samples (~1.5 mg each) onto a 12% Bis-Tris gel. Samples were electrophoresed into the gel, and the gel was fixed with the same Fixative Solution used with the Pierce Silver Stain Kit for Mass Spectrometry (ThermoFisher, 24600). The gel then underwent in-gel trypsin digestion, followed by peptide desalting and LC-MS/MS analysis for protein identification using an Easy-nLC 1200 coupled to an Orbitrap Fusion Lumos mass spectrometer. We used label-free spectral counting to quantitate protein abundance. MaxQuant software was used to process data and to search against the UniProt human protein database. The LC-MS/MS was performed on three biological replicates per group by Drs. Mengmeng Zhu and Guoan Zhang in the Weill Cornell Medicine LC-MS/MS Core.
5
Immunoprecipitation and Mass Spectrometry Analysis
6
Recombinant ΔN-SNM1A Interaction with CSB
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Recombinant ΔN-SNM1A (500 ng) was incubated with or without HA-CSB (500 ng) in the presence of α-HA magnetic beads (Thermo Fisher Scientific, Rockford, IL, USA) in a 500 μl reaction containing 20 mM HEPES pH 7.9, 4 mM MgCl2, 0.05 mM ATP, 40 μg/ml bovine serum albumin (BSA) and 1 mM DTT at 4°C for 2 h. The beads and associated material were captured on a magnetic stand via a 1-min incubation, and washed three times with 20 mM HEPES pH 7.9, 4 mM MgCl2, 1 mM DTT and 0.1% Nonidet P-40. The bead-bound material was suspended in 2× sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) loading dye and incubated at 95°C for 5 min. Proteins were resolved on an 8% Tris-glycine-SDS polyacrylamide gel and detected using the Pierce Silver Stain Kit for Mass Spectrometry (Thermo Fisher Scientific).
7
Identification of Immunoprecipitated Proteins
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Immunoprecipitated proteins were resolved by SDS-PAGE (Nu-PAGE, 4–12% Bis–Tris precast gels, Invitrogen) and stained with Coomassie or silver-stained using the Pierce Silver Stain kit for Mass Spectrometry (Thermo Fisher Scientific). Proteins were detected by western blotting using an anti-RNAP β subunit antibody [clone 8RB13] (BioLegend) or anti-σ70 antibody [clone 2G10] (BioLegend), and a HRP-labeled anti-mouse IgG antibody (Sigma-Aldrich). The identity of the protein bands was determined by MALDI-TOF mass spectrometry as described previously (52 (link)).
8
Immunoprecipitation and Immunoblotting Analysis
9
SDS-PAGE and Western Blot Analysis
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Cells were lysed in SDS lysis buffer (50 mM Tris-HCl pH7.5, 100 mM NaCl, 1 mM EDTA, 1% SDS, 10 mM NaF, 10 mM β-glycerophosphate, 2 mM Na3VO4). Fractions from the vesicle isolation, or cell lysates were subjected to SDS-PAGE, followed by transfer to Immobilon-P PVDF membranes (Millipore). Membranes were blocked in 1% BSA/TBS containing 0.1% Tween20 for 30 min, and treated with primary antibodies in blocking buffer overnight at 4°C, followed by treatment with HRP-conjugated secondary antibodies (Dako) for 1 h. Bands were visualized using Chemi-Lumi One Super or Chemi-Lumi One Ultra (Nacalai Tesque, 02230–30 and 11644–40) according to the manufacture’s protocol, and images were obtained using an Luminograph I quantitative Cooled CCD camera detection system (ATTO) or an Amersham™ ImageQuant™ 800 (Cytiva) as unsaturated 16-bit TIFF images. Silver staining of gels were performed using Pierce™ Silver Stain for Mass Spectrometry kit (Thermo Fisher, 24600).
10
Identification of ET-interacting proteins
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Putative ET-interaction partners were co-immunoprecipitated from S2 cell protein lysates using Anti-V5 Agarose Affinity Gel (Sigma, #A7345, Israel) according to the manufacturer’s instructions. The following lysates were used: S2 cells (neg. ctrl 1), empty pMT-vector (neg. ctrl 2), pMT-ET-V5 and pMT-ET-V5 + pMT-upd1-myc. 900 µl of each lysate, containing approximately 2.5 mg of protein, was incubated overnight with the Anti-V5 Agarose Affinity Gel, after which the complexes were washed 6 x 10 minutes with PBS containing protease and phosphatase inhibitor cocktail. All incubations and treatments were carried out at 4°C or on ice. Finally, loading buffer was added on the washed affinity gel containing protein complexes and the samples were boiled to release the proteins. Purified protein complexes were analyzed by SDS-PAGE and silver-staining.
For SDS-PAGE, Novex 10% NuPAGE Bis-Tris (Life Technologies, #NP0301BOX, USA) gels were used. Precision Plus Protein™ Dual Xtra Standard (Bio-Rad, #161-0377, USA) was used as a marker. The electrophoresis was carried out for 45 min using MOPS buffer and NuPAGE Gel program (200 V, 120 mA, 25 W). To stain the protein bands on the gel, the Pierce® Silver Stain for Mass Spectrometry kit (Thermo Scientific, #24600, USA) was used according to the manufacturer’s instructions.
For SDS-PAGE, Novex 10% NuPAGE Bis-Tris (Life Technologies, #NP0301BOX, USA) gels were used. Precision Plus Protein™ Dual Xtra Standard (Bio-Rad, #161-0377, USA) was used as a marker. The electrophoresis was carried out for 45 min using MOPS buffer and NuPAGE Gel program (200 V, 120 mA, 25 W). To stain the protein bands on the gel, the Pierce® Silver Stain for Mass Spectrometry kit (Thermo Scientific, #24600, USA) was used according to the manufacturer’s instructions.
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