Snap i d 2.0 protein detection system

Manufactured by Merck Group

Sourced in United States, Germany

The SNAP i.d.® 2.0 Protein Detection System is a lab equipment product designed for efficient protein detection. It provides a streamlined workflow for Western blotting procedures.

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Lab products found in correlation

Anti β actin monoclonal antibody, by Merck Group (3 mentions) Pvdf membrane, by Merck Group (3 mentions) Glass bead, by Merck Group (2 mentions) Anti cd38 polyclonal antibody, by Santa Cruz Biotechnology (2 mentions) T per protein extraction reagent, by Thermo Fisher Scientific (2 mentions) Ecl prime western blotting detection kit, by GE Healthcare (2 mentions) Quick start bradford protein assay, by Bio-Rad (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Ab290, by Abcam (1 mentions) Rneasy kit, by Qiagen (1 mentions) Mini protean tetra vertical electrophoresis cell, by Bio-Rad (1 mentions) Trans blot turbo mini pvdf transfer pack, by Bio-Rad (1 mentions) Picoscope, by Ushio (1 mentions) Hrp conjugated rabbit anti mouse igg, by Agilent Technologies (1 mentions) Azurespot analysis software, by Azure Biosystems (1 mentions) Iblottm dry blotting system, by Thermo Fisher Scientific (1 mentions) Azure c400 system, by Azure Biosystems (1 mentions) Mini protean tgx precast gel, by Bio-Rad (1 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Mouse monoclonal anti adar1 antibody 15.8.6, by Santa Cruz Biotechnology (1 mentions)

17 protocols using snap i d 2.0 protein detection system

Quantitative Transcriptomic Analysis of GCTB

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Purified total RNA (RNeasy kit, Qiagen) from 22 GCTB fresh frozen biopsies and 13 cell line samples (Supplementary Table S1) were used in the nCounter hybridization-based RNA transcript quantification technology (Nanostring). The validation set contained 12 samples not used in the test set. We had 39 genes designed for the nCounter gene expression custom codeset to validate the identified splicing defects and alternative starting sites seen in the six GCTB cell line sample set.
Western blot analysis was performed with standard PAGE and submarine protein transfer system (BioRad), and detection with the SNAP i.d. 2.0 protein detection system (Merck Millipore). Antibodies used were anti-GFP (ab290, Abcam), and anti-hnRNPA1L2 (ab180124, Abcam).

Lim J., Park J.H., Baude A., Yoo Y., Lee Y.K., Schmidt C.R., Park J.B., Fellenberg J., Zustin J., Haller F., Krücken I., Kang H.G., Park Y.J., Plass C, & Lindroth A.M. (2017). The histone variant H3.3 G34W substitution in giant cell tumor of the bone link chromatin and RNA processing. Scientific Reports, 7, 13459.

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Sperm Tyrosine Phosphorylation Assay

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Sperm were incubated in a 100-μL drop of cTYH for 0 or 60 min. After incubation, the concentration of each sperm suspension was measured using a PiCOSCOPE® (USHIO, Japan) and equalized. Aliquots of the sperm suspension were collected by centrifugation at 14,000 × g for 5 min. Subsequent to washing with 1 mL of phosphate-buffered saline, the sperm pellet was resuspended in Laemmli sample buffer and 2-mercaptoethanol and boiled for 5 min. After disruption of sperm sample by ultrasonication for 1 min, SDS-PAGE was performed using Mini-PROTEAN® Tetra Vertical Electrophoresis Cell (Bio-Rad, USA), and proteins were transferred to a Trans-Blot® Turbo Mini PVDF Transfer Pack (Bio-Rad). Western blot analysis was performed using the SNAP i.d.® 2.0 Protein Detection System (Merck Millipore, Germany). The protein-transferred membrane was blocked with PO Noise Cancelling Reagents for Phosphoprotein Detection using chemiluminescence or fluorescence techniques (Bløk; Merck Millipore) at room temperature. After removing the blocking solution, the membrane was immunoblotted with a monoclonal antibody against phosphotyrosine (4G10® Platinum, Anti-Phosphotyrosine Antibody; Merck Millipore) for 10 min at room temperature and horseradish peroxidase-conjugated secondary antibodies for 10 min at room temperature. α-Tubulin was used as the internal control.

Yoshimoto H., Takeo T, & Nakagata N. (2017). Dimethyl sulfoxide and quercetin prolong the survival, motility, and fertility of cold-stored mouse sperm for 10 days. Biology of Reproduction, 97(6), 883-891.

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Recombinant Fusion Antigen Expression in Lactobacillus

Cited 1 time

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To analyze expression of recombinant fusion antigen in L. plantarum, cells from a 50 mL culture were harvested 3 h after induction (25 (link), 43 (link)) and resuspended in 500 μL PBS. Bacterial protein extracts were prepared by disruption in FastPrep tubes containing 1.5 g of glass beads (size ≤ 106 μm; Sigma-Aldrich), using a FastPrep® FP120 Cell Disrupter with a shaking speed of 6.5 m/s for 45 s. After 5 min incubation on ice the shaking process was repeated. The glass beads were removed by sedimentation and the protein extracts were transferred to a new tube. Proteins were separated by SDS-polyacrylamide gel electrophoresis using 10% Mini-Protean TGX Precast gels (BioRad) and transferred to a nitrocellulose membrane using the iBlot^TM Dry Blotting System (Invitrogen). The proteins were detected using the SNAP i.d.® 2.0 Protein Detection System (Merck) using a specific monoclonal mouse anti-ESAT-6 antibody (Abcam) diluted 1:15000 and, subsequently, a polyclonal HRP-conjugated rabbit anti-mouse IgG (DAKO), diluted 1:7500. Proteins were visualized using the SuperSignal™ West Pico Chemiluminescent Substrate (Termo Fisher Scientific) and signals were documented using an Azure c400 system and AzureSpot Analysis Software (Azure Biosystems), following the manufacturer's instructions.

Kuczkowska K., Copland A., Øverland L., Mathiesen G., Tran A.C., Paul M.J., Eijsink V.G, & Reljic R. (2019). Inactivated Lactobacillus plantarum Carrying a Surface-Displayed Ag85B-ESAT-6 Fusion Antigen as a Booster Vaccine Against Mycobacterium tuberculosis Infection. Frontiers in Immunology, 10, 1588.

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Immunoblotting of ADAR Enzymes in Mouse Tissues

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Tissue lysates from mouse cerebral cortex and spleen were prepared and stored at −80°C until use as described previously (Miyake et al. 2016 (link)). Lysates were then separated using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules) and immunoblotted with primary antibodies using a SNAP i.d 2.0 Protein Detection System (Merck Millipore) as previously described (Nakahama et al. 2018 (link)). The primary antibodies used were as follows: mouse monoclonal anti-ADAR1 antibody (15.8.6; Santa Cruz Biotechnology), mouse monoclonal anti-ADAR2 antibody (1.3.1; Santa Cruz Biotechnology), and mouse monoclonal anti-GAPDH (M171-3; MBL).

Costa Cruz P.H., Kato Y., Nakahama T., Shibuya T, & Kawahara Y. (2020). A comparative analysis of ADAR mutant mice reveals site-specific regulation of RNA editing. RNA, 26(4), 454-469.

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Western Blot Analysis of LC3B

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Collected cell pellets were extracted in RIPA buffer (20 mM HEPES, pH 7.0, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 10 mM β-Glycerophosphate, 1 mM Na₃VO₄, and 5 mM NaF) and protein concentrations were determined with the BCA protein assay kit (Thermo Fisher Scientific, Darmstadt, Germany; #23227). The proteins were separated on 12% gels by electrophoresis and were transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA; ISEQ00010). The membranes were blocked in 0.5% BSA in Tris-buffered saline with Tween-20 (TBS-T) and were probed with: anti-LC3B (Cell signaling, Danvers, MA, USA; #3868) and anti-α-tubulin (Sigma-Aldrich, Saint Louis, MO, USA; T5168), followed by secondary HRP-antibodies; anti-mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA; sc-2005), and anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA; sc-2004). The blocking and probing were performed using SNAP i.d. 2.0 Protein Detection System (Merck Millipore, Billerica, MA, USA). Proteins were detected using Pierce ECL chemiluminescence kit (Thermo Fisher Scientific, Darmstadt, Germany; #32132). The bands were quantified by ImageJ (National Institutes of Health, Bethesda, MD, USA).

Kim H., Choi S.Y., Lim J., Lindroth A.M, & Park Y.J. (2020). EHMT2 Inhibition Induces Cell Death in Human Non-Small Cell Lung Cancer by Altering the Cholesterol Biosynthesis Pathway. International Journal of Molecular Sciences, 21(3), 1002.

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Immunoblot Analysis of H9c2 Cardiomyocyte Proteins

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H9c2 cardiomyocyte extract (5 × 10⁵ cells) was subjected to immunoblot analysis as previously described [46 (link),64 (link)], using an anti-Cd38 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) raised against a peptide fragment of mouse Cd38 (residues 279–301 in [46 (link)]), anti-Ryr2 polyclonal antibody (PeproTech, Canbury, NJ, USA) [67 (link)], anti-Fkbp12.6 monoclonal antibody (Santa Cruz Biotechnology) raised against the amino acids 38–108 of human/rat/mouse FKBP12.6, anti-Pten monoclonal antibody raised against the full-length human PTEN protein (Abcam, Cambridge, UK), and anti-β-actin monoclonal antibody (Sigma, St. Louis, MO, USA) raised against Ac-Asp-Asp-Asp-Ile-Ala-Ala-Leu-Val-Ile-Asp-Asn-Gly-Ser-Gly-Lys. A SNAP id^® 2.0 Protein Detection System (Merck Millipore, Burlington, MA, USA) was used for the analysis. The band intensities were analyzed using ImageJ software (National Institute of Health, Bethesda, MD, USA), as previously described [35 (link),46 (link),64 (link),82 (link),83 (link)].

Takasawa S., Makino M., Uchiyama T., Yamauchi A., Sakuramoto-Tsuchida S., Itaya-Hironaka A., Takeda Y., Asai K., Shobatake R, & Ota H. (2022). Downregulation of the Cd38-Cyclic ADP-Ribose Signaling in Cardiomyocytes by Intermittent Hypoxia via Pten Upregulation. International Journal of Molecular Sciences, 23(15), 8782.

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Quantifying CCL3Gag Expression in Bacterial Cells

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To analyze CCL3Gag expression, bacterial cells were harvested from 50 ml of cultures, resuspended in 1 ml PBS and added to FastPrep tubes containing glass beads (Sigma-Aldrich). Cell-free protein extracts were prepared by disruption in a FastPrep^® FP120 Cell Disrupter by shaking at a speed of 6.5 m/s for 45 s. Cell debris was removed by centrifugation at 12,000×g, 4 °C, for 2 min. Proteins were separated by SDS–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane using the iBlot™ Dry Blotting System (Invitrogen). Proteins were detected using the SNAP i.d.^® 2.0 Protein Detection System (Merck kGaA Darmstadt, Germany) using a specific polyclonal goat anti-CCL3 antibody (R&D Systems, BAF450), 1:10,000 and, subsequently, a polyclonal rabbit anti-goat HRP-conjugated (Abcam) antibody, diluted 1:5000.

Kuczkowska K., Mathiesen G., Eijsink V.G, & Øynebråten I. (2015). Lactobacillus plantarum displaying CCL3 chemokine in fusion with HIV-1 Gag derived antigen causes increased recruitment of T cells. Microbial Cell Factories, 14, 169.

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Protein Extraction and Western Blotting

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The proteins were extracted from different cells using T-PER Protein Extraction Reagent (Thermo Fisher Scientific Inc.). Protein concentrations were quantified with Quick Start Bradford Protein Assay (Bio-Rad). Denatured proteins (20 µg) were then analyzed using 10% e-PAGELs (ATTO Corporation, Tokyo, Japan). After electrophoresis, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore). The membrane was blocked with 0.5% skim milk in phosphate buffered saline solution (pH 7.6) containing 0.1% Tween-20 (PBS/T), and then SNAP i.d. 2.0 Protein Detection system (Merck Millipore). The membrane was subsequently stripped and blotted with polyclonal rabbit anti-turboGFP antibody (Evrogen JSC, Moscow, Russia) (1:2,000 dilutions) and polyclonal rabbit anti-ACTIN (Sigma-Merck Millipore) (1:2,000 dilutions) to control for protein loading. HRP-conjugated secondary antibodies (Cell Signaling, Tokyo, Japan) were used at a dilution of 1:2,000 and developed using the ECL Prime Western Blotting Detection Kit (GE Healthcare, Buckinghamshire, UK). Exposure for chemiluminescent samples or membrane analysis for the blots was performed by LAS4000 (GE Healthcare).

Okumura K., Saito M., Yoshizawa Y., Munakata H., Isogai E., Miura I., Wakana S., Yamaguchi M., Shitara H., Taya C., Karaplis A.C., Kominami R, & Wakabayashi Y. (2017). The parathyroid hormone regulates skin tumour susceptibility in mice. Scientific Reports, 7, 11208.

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Protein Extraction and Western Blot Analysis

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Proteins were extracted from different cells using T‐PER Protein Extraction Reagent (Thermo). Protein concentrations were quantified by the Quick Start Bradford Protein Assay (Bio‐Rad). Denatured proteins (Mouse normal skins: 60 μg) were then analyzed using 15% e‐PAGELs (ATTO). After electrophoresis, these were transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck Millipore). The membrane was blocked with 0.5% skimmed milk or 1% BSA in phosphate‐buffered saline solution (pH 7.6) containing 0.1% Tween‐20 (PBS/T) and then the SNAPi.d. 2.0 Protein Detection system (Merck Millipore). Primary antibodies were as follows: anti‐Cenp‐50/U (anti‐rabbit polyclonal),¹⁷ anti‐Actin (Sigma‐Merck Millipore). HRP‐conjugated secondary antibodies were used at a dilution of 1:2000 and developed using the ECL Prime Western Blotting Detection Kit (GE Healthcare). Exposure for chemiluminescent samples or membrane analysis for the blots was performed with a LAS 4000 system (GE Healthcare).

Saito M., Kagawa N., Okumura K., Munakata H., Isogai E., Fukagawa T, & Wakabayashi Y. (2020). CENP‐50 is required for papilloma development in the two‐stage skin carcinogenesis model. Cancer Science, 111(8), 2850-2860.

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Immunoblot Analysis of CD38 Protein

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The immunoblot analysis was performed using an As4.1 cell extract (5 × 10⁵ cells), as described in previous studies [10 (link),52 (link),60 (link)], using an anti-Cd38 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) raised against a peptide fragment of mouse Cd38 (residues 279–301 in [61 (link)]), an anti-β-actin monoclonal antibody (Sigma, St. Louis, MO, USA) raised against Ac-Asp-Asp-Asp-Ile-Ala-Ala-Leu-Val-Ile-Asp-Asn-Gly-Ser-Gly-Lys, and a SNAP id^® 2.0 Protein Detection System (Merck Millipore, Burlington, MA, USA). The band intensities were analyzed using ImageJ software (National Institute of Health, Bethesda, MD, USA), as previously described [52 (link),62 (link),63 (link)].

Takeda Y., Itaya-Hironaka A., Yamauchi A., Makino M., Sakuramoto-Tsuchida S., Ota H., Kawaguchi R, & Takasawa S. (2021). Intermittent Hypoxia Upregulates the Renin and Cd38 mRNAs in Renin-Producing Cells via the Downregulation of miR-203. International Journal of Molecular Sciences, 22(18), 10127.

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