Sa1 200

Manufactured by Thermo Fisher Scientific

The SA1-200 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed to perform a core function within the laboratory setting. Detailed specifications and intended use are not available in this factual and unbiased description.

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4 protocols using sa1 200

Protein Immunoprecipitation and Western Blot

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ECs were placed on ice before being lysed at 4 °C in lysis buffer (25 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM MgCl₂, 1 mM Na₃VO₄, 0.5 mM EGTA, 1% Triton X-100, 5% glycerol, supplemented with Halt^TM protease inhibitors) and cleared by centrifugation at 12,000 × g for 20 min at 4 °C. Supernatant proteins were then quantified using the BCA assay and equivalent protein concentrations (800 µg) were immunoprecipitated with Dynabeads^TM Protein G (Invitrogen) coupled to a primary antibody at 4 °C. Immunoprecipitated proteins, alongside a control IgG sample (Fisher Scientific: SA1-200, RRID: AB_325994), were separated by SDS-PAGE and subjected to Western blot analysis.

Benwell C.J., Johnson R.T., Taylor J.A., Lambert J, & Robinson S.D. (2024). A proteomics approach to isolating neuropilin-dependent α5 integrin trafficking pathways: neuropilin 1 and 2 co-traffic α5 integrin through endosomal p120RasGAP to promote polarised fibronectin fibrillogenesis in endothelial cells. Communications Biology, 7, 629.

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Western Blot Analysis of rAbcc6 Protein

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Cell lysates were prepared as previously described (22 ). Five (5 ) µg of total protein was separated on a 7.5% SDS-polyacrylamide gel (Bio-Rad) and transferred to a PVDF membrane using the Trans Blot Turbo system (Bio-Rad). Wild type and mutant rAbcc6 was detected with the polyclonal K14 rabbit anti-rAbcc6 antibody (kind gift of Dr. Bruno Stieger) diluted 1:3000 and HRP-conjugated donkey anti-rabbit secondary antibody (1:5000, SA1200, Fisher Scientific). His¹⁰ tagged rAbcc6 fusion proteins were also detected with the anti-His⁶ mouse monoclonal antibody (1:250, MA1–21315, Thermo Fisher), followed by incubation with HRP-conjugated anti-mouse secondary antibody (1:5000). Anti-α-tubulin (1:1000, Sc-23948, Santa-Cruz Biothechnology) was used as loading control, with HRP-conjugated polyclonal rabbit anti-mouse IgG employed as secondary antibody (1:5000, P0161, Dako). Antibody binding was visualized by ECL (Pierce Western blotting substrate, Thermo Scientific).

Szeri F., Niaziorimi F., Donnelly S., Orndorff J, & van de Wetering K. (2020). Generation of fully functional fluorescent fusion proteins to gain insights into ABCC6 biology. FEBS letters, 595(6), 799-810.

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Western Blot Analysis of Cx43 Expression

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Cells were lysed in radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 5 mM EDTA and protease/phosphatase inhibitors cocktail (all from Thermo Fisher Scientific) for 30 min on ice. Western blotting was conducted as described previously [18 (link)], loading 30 µg of total proteins per well. For protein detection anti-Cx43 (dilution 1:500; catalog no. C6219, Sigma-Aldrich), donkey anti-rabbit HRP-conjugated (dilution 1:5000; SA1-200, Thermo Fisher Scientific) and anti-β-actin IgG1 HRP-conjugated (dilution 1:10,000; SC-47778, Santa Cruz Biotechnology) antibodies were used. The membranes were revealed using the SuperSignal^® West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific) according to manufacturer′s instructions and analyzed in an ImageQuant LAS 500 (GE Healthcare, Uppsala, Sweden). Images were analyzed using ImageJ software (National Institutes of Health).

Hofmann F., Navarrete M., Álvarez J., Guerrero I., Gleisner M.A., Tittarelli A, & Salazar-Onfray F. (2019). Cx43-Gap Junctions Accumulate at the Cytotoxic Immunological Synapse Enabling Cytotoxic T Lymphocyte Melanoma Cell Killing. International Journal of Molecular Sciences, 20(18), 4509.

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Protein Expression Analysis by Western Blot

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Protein samples were isolated and further separated by 4%–12% SDS-PAGE gel (NW04125BOX, Thermo Fisher Scientific). After protein transfer, immunoblots were performed using specific primary antibodies and HRP-conjugated secondary antibodies. Primary antibodies are as follows: pannexin-1 (D9M1C) rabbit mAb (91137S, 1:1000, Cell Signaling Technology), anti-NLRP3/NALP3, mAb (Cryo-2) (AG-20B-0014-C100, 1:1000, AdipoGen), IL-1β polyclonal antibody (PA420B, 1:1000, Invitrogen), cyclophilin B polyclonal antibody (PA1027A, 1:2000, Invitrogen), and GAPDH antibody (MA5-15738, 1:2000, Invitrogen). Secondary antibodies are as follows: donkey anti-mouse IgG secondary antibody (SA1-100, 1:2000, Thermo Fisher Scientific) and donkey anti-rabbit IgG secondary antibody (SA1-200, 1:2000, Thermo Fisher Scientific). Signals were detected with ECL Western Blotting Substrate (32106, Thermo Fisher Scientific).

Zhang S., Yuan B., Lam J.H., Zhou J., Zhou X., Ramos-Mandujano G., Tian X., Liu Y., Han R., Li Y., Gao X., Li M, & Yang M. (2021). Structure of the full-length human Pannexin1 channel and insights into its role in pyroptosis. Cell Discovery, 7, 30.

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