For processing of lipocalins with active cathepsin S, 200 μg of each lipocalin was incubated for 24 hours at 37°C with 15 μg of active human recombinant cathepsin S (BioVision, Milpitas, Calif) in 100 μL of sterile degradation buffer containing 100 mmol/L NaCl, 5 mmol/L EDTA, 10 mmol/L sodium acetate (pH 5), and 2 mmol/L dithiotreitol.26 (link),27 Samples were stored at −20°C and used for Ca2+ mobilization assays. We used high-concentration lipocalin protein solutions to omit buffer exchange of proteins with cathepsin S digestion buffer. In addition, digests of Lcn-1 and Can f 1 were withdrawn after 1, 5, 10, 24, and 48 hours and applied on a 12% Bis-Tris gel. As a control, the lipocalins were incubated for 48 hours at 37°C in digestion buffer without cathepsin S. The gels were cut into 2 parts between the 10- and 15-kDa markers. The upper parts with the larger proteins were stained with InstantBlue (Expedeon, Harston, United Kingdom), and the lower part was silver stained according to the manufacturer’s protocol with the ProteoSilver Kit (Sigma-Aldrich, St Louis, Mo).
Proteosilver kit
Manufactured by Merck Group
Sourced in Sao Tome and Principe, United States
The ProteoSilver Kit is a laboratory product designed for the detection and visualization of proteins in polyacrylamide gels. It provides a sensitive and reliable method for staining proteins after electrophoretic separation, enabling their identification and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Active human recombinant cathepsin s, by Abcam (2 mentions)
Instantblue, by Abcam (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Hydrogen peroxide h2o2, by Merck Group (1 mentions)
Glycerol, by Merck Group (1 mentions)
Anti myc agarose, by Merck Group (1 mentions)
Trypsin kit, by Thermo Fisher Scientific (1 mentions)
Anti flag agarose, by Merck Group (1 mentions)
Anti flag m2 agarose, by Merck Group (1 mentions)
Sds page gel, by Thermo Fisher Scientific (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Merck Group (1 mentions)
Supersignal west dura chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
8 protocols using proteosilver kit
1
Cathepsin S Degradation of Lipocalins
2
Antibody Detection Using Recombinant HAV VP1
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Detergents (Tween 20 and sodium dodecyl sulfate (SDS)), bovine serum albumin (BSA), ProteoSilver™ Kit, diaminobenzidine, β-mercaptoethanol, and horseradish peroxidase-conjugated goat anti-human IgM antibody (IgM HP) were acquired from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The HAV viral protein 1 (HAV VP1, recombinant) was acquired from Meridian Life Science (Memphis, Tennessee, USA). Nitrocellulose (0.2 µm), Precision Plus Protein™ Kaleidoscope™ Prestained Protein Standards and electrophoresis reagents were acquired from Bio-Rad Laboratories Inc. (US). Hydrogen peroxide (H2O2) and glycerol were purchased from Merck Millipore Corporation (Darmstadt, Germany). Amine coupling agents (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS)) and ethanolamide were purchased from Cytiva (Piscataway, NJ, USA). A carboxylated gold sensor chip (COOH5) was acquired from FortéBio-Sartorius BioAnalytical Instruments (Fremont, CA, USA). All other reagents were of analytical grade or better.
3
Cathepsin S Degradation of Lipocalins
4
Investigating Smad4-HDCA1 Interaction in IDD
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The degenerative IVD tissue (0.1 g) from an IDD patient under Pfirrmann grade IV was homogenized in 1 mL RIPA buffer containing the protease inhibitor. The supernatant of the cell lysate was immunoprecipitated using anti-Smad4- and IgG-coupled protein A beads (Santa Cruz Biotechnology, Shanghai, China; #sc-2001). The enriched proteins were rinsed five times with PBST buffer and then loaded onto a 12% SDS-PAGE gel for separation, followed by staining with the ProteoSilver Kit (Sigma-Aldrich; #PROTSIL2). The positive bands were cut into small pieces and then digested using the Trypsin Kit (Thermo Fisher Scientific; #60109101). The eluted proteins were subjected to MS analysis.
The co-IP assay was performed as described previously 25 (link). In brief, different combinations of plasmids, including pCDNA3-2×Flag + pCDNA3-Myc, pCDNA3-2×Flag + pCDNA3-Myc-HDCA1, pCDNA3-2×Flag-Smad4 + pCDNA3-Myc-HDCA1, and pCDNA3-2×Flag-Smad4 + pCDNA3-Myc were co-transfected into HNPC cells. After 48 hours of transfection, cells were subjected to the IP procedure using anti-Flag agarose (Sigma-Aldrich; #A4596) and anti-Myc-agarose (Sigma-Aldrich; #A7470). The enriched protein complexes were probed with anti-Flag (Abcam; #ab125243) and anti-Myc (Abcam; #ab32).
The co-IP assay was performed as described previously 25 (link). In brief, different combinations of plasmids, including pCDNA3-2×Flag + pCDNA3-Myc, pCDNA3-2×Flag + pCDNA3-Myc-HDCA1, pCDNA3-2×Flag-Smad4 + pCDNA3-Myc-HDCA1, and pCDNA3-2×Flag-Smad4 + pCDNA3-Myc were co-transfected into HNPC cells. After 48 hours of transfection, cells were subjected to the IP procedure using anti-Flag agarose (Sigma-Aldrich; #A4596) and anti-Myc-agarose (Sigma-Aldrich; #A7470). The enriched protein complexes were probed with anti-Flag (Abcam; #ab125243) and anti-Myc (Abcam; #ab32).
5
Identifying GmLMM1-Interacting Proteins
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To identify GmLMM1‐interacting proteins, the coding sequences of GmLMM1 were amplified by PCR and cloned into the pUC19‐35S‐FLAG‐RBS vector. The GmLMM1‐FLAG was transfected into WT (Col‐0) Arabidopsis protoplasts and incubated overnight. Total protein was extracted with IP buffer (50 mM HEPES [pH 7.5], 50 mM NaCl, 10 mM EDTA, 0.2% Triton X‐100, 0.1 mg/ml Dextran (Sigma), proteinase inhibitor cocktail) and incubated with anti‐FLAG M2 agarose (Sigma) for 4 h. The agarose was washed twice with buffer A (50 mM HEPES [pH 7.5], 50 mM NaCl, 10 mM EDTA, 0.1% Triton X‐100) and twice with buffer B (50 mM HEPES [pH 7.5], 150 mM NaCl, 10 mM EDTA, 0.1% Triton X‐100), and eluted with 3 × FLAG peptide (Sigma) for 40 min. The immunoprecipitates were separated on a 10% SDS–PAGE gel (Invitrogen) and stained with the ProteoSilver Kit (Sigma). The gel was de‐stained and digested in‐gel with trypsin (10 ng/μl trypsin, 50 mM ammonium bicarbonate [pH 8.0]) at 37°C overnight and then subjected to mass spectrometric analysis by PTM Bio (Hangzhou, China) as previously described (Li et al, 2014 ).
6
RNA-Binding Protein Immunoprecipitation
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The gene perturbation‐treated cells (5 million) were lysed with 500 ml of lysis buffer (87787, Thermo; 0.5 mM PMSF; and protease inhibitor cocktail, Roche). Similar to the RIP assay, the samples were also pre‐cleared with Dynabeads Protein G, and 1/15 of them were saved as a reference for input. Meanwhile, Dynabeads Protein G was mixed with a specific antibody (c‐Src or IgG) on a shaker for 2 h (4 °C). After that, the samples were incubated overnight in the antibody‐bead slurry. Both buffer I (50 mM Tris pH 7.4, 2 mM RVC, 150 mM NaCl, 0.5 mM PMSF, 0.5% NP‐40, protease inhibitor cocktail; three times, 5 min) and buffer II (50 mM Tris pH7.4, 2 mM RVC, 0.5 mM PMSF, 350 mM NaCl, 1% NP‐40, protease inhibitor cocktail; three times, 5 min) were used to wash the samples the following day. Western blotting or subsequent mass spectrometry analysis was performed to examine the final samples.
For mass spectrometry analysis, the proteins were separated by SDS‐PAGE, and the ProteoSilver Kit (Sigma‐Aldrich, St. Louis, Mo, USA) was used for silver staining. Finally, the bands were collected for mass spectrometry.
For mass spectrometry analysis, the proteins were separated by SDS‐PAGE, and the ProteoSilver Kit (Sigma‐Aldrich, St. Louis, Mo, USA) was used for silver staining. Finally, the bands were collected for mass spectrometry.
7
Validation of Anti-Rhg1 and Anti-SNAP Antibodies
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Generation and validation of rabbit polyclonal antibodies raised against Rhg1 α-SNAP LC , Rhg1 α-SNAP HC , and α-SNAP WT was previously described in Bayless et al. (2016) (link). Tissue preparation and immunoblots were performed essentially as in Bayless et al. (2016 (link)Bayless et al. ( , 2018)) (link). Bradford assays were performed on each leaf or root extract supernatant and equal amount of OD 595 total protein were loaded onto SDS-PAGE gels. Immunoblots for α-SNAP and NSF were incubated overnight at 4 ˚C in 5% nonfat dry milk TBS-T (50 mM Tris, 150 mM NaCl, 0.05% Tween 20) at 1:1000. Secondary horseradish peroxidase conjugated goat antirabbit IgG (Sigma) was added at 1:10,000 and incubated for 1 h at room temperature on a platform shaker followed by five washes with TBS-T. Chemiluminescence signal detection was performed with SuperSignal West Dura chemiluminescent substrate (Thermo Scientific) and developed using a Chemi Doc MP chemiluminescent imager (Bio-Rad).
For antibody sensitivity assays, purified recombinant α-SNAP was serially fivefold diluted to concentrations of 800, 160, and 32 pg. The proteins were then loaded onto an SDS-PAGE gel and immunoblots performed as above. To confirm loading of gel lanes, the gel was stained using ProteoSilver Kit (Sigma) according to manufacturer's instruction.
For antibody sensitivity assays, purified recombinant α-SNAP was serially fivefold diluted to concentrations of 800, 160, and 32 pg. The proteins were then loaded onto an SDS-PAGE gel and immunoblots performed as above. To confirm loading of gel lanes, the gel was stained using ProteoSilver Kit (Sigma) according to manufacturer's instruction.
8
α-SNAP Binding to NSF Assay
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In vitro binding assays were performed as previously described (Bayless et al., 2016) (link). In brief, 20 μg of the specified recombinant α-SNAP was added to the bottom of a 1.5 mL polypropylene tube, unbound α-SNAP was removed with wash buffer, and 20 μg of either NSF or NSF RAN07 in NSF binding buffer (20 mM HEPES, 2 mM EDTA, 100 mM KCl, 500 μM ATP, 1 mM DTT, 1% (w/v) PEG 4000) was added and incubated on ice for 10 min. The solution was then removed and excess NSF was removed by washing twice with NSF binding buffer. Samples were subsequently boiled in 1× SDS loading buffer and separated on a 10% Bis-Tris SDS-PAGE gel. To detect bound NSF, the gel was stained using the Pro-teoSilver Kit (Sigma) according to manufacturer's instruction and quantified relative to α-SNAP abundance by densitometric analysis using ImageJ.
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