Rpn2232

Manufactured by Bio-Rad

The RPN2232 is a lab equipment product manufactured by Bio-Rad. It is a core piece of equipment used for essential laboratory functions. No further details can be provided in an unbiased and factual manner.

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Lab products found in correlation

Lc2001, by Thermo Fisher Scientific (2 mentions) Chemidoc touch, by Bio-Rad (2 mentions) Na931, by Cytiva (2 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions) Sc 271527, by Cytiva (2 mentions) Mca4739p, by Bio-Rad (2 mentions) Gel doc xr imager, by Bio-Rad (1 mentions) Chemidoc, by Bio-Rad (1 mentions) Hpv16 e6 e6 6f4, by Euromedex (1 mentions) Igg anti mouse igg h l hrpo, by Dianova (1 mentions) β actin ac 74, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions)

4 protocols using rpn2232

Western Blot Analysis of MX2 Protein

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Cells collected on the day of HIV infection were lysed in RIPA buffer (50 mM Tris pH 8.0, 0.1% SDS, 1% Triton-X, 150 mM NaCl, 1% deoxycholic acid, 2 mM PMSF). Cell extracts were prepared in 4X LDS Buffer (Invitrogen #NP007) and 2X MES SDS Running Buffer (Invitrogen #NP002). After boiling at 100C for 5 min, 20 μg of the prepared samples were loaded into a NuPAGE 4–12% Bis-Tris Gel (Invitrogen #NP0321) and resolved. Following transfer onto a nitrocellulose membrane (Invitrogen #LC2001), blots were blocked in blocking buffer (PBS, 0.1% Tween, 5% milk) for 1 h, incubated with the MX2 primary antibody (Santa Cruz Biotech #sc-271527; 1:100) overnight, probed with secondary antibody (Cytiva #NA931, 1:1000) for 1 h, and visualized by chemiluminescence (Cytiva #RPN2232) on a Bio-Rad ChemiDoc Touch. Membranes were washed in PBST (PBS, 0.1% Tween) in between each step. After imaging the MX2 stain, membranes were stripped (ThemoFisher #46430), washed, probed with GAPDH:HRP (Bio-Rad #MCA4739P, 1:1000) for 1 h, and imaged with chemiluminescence.

Itell H.L., Humes D, & Overbaugh J. (2023). Several cell-intrinsic effectors drive type I interferon-mediated restriction of HIV-1 in primary CD4+ T cells. bioRxiv.

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Western Blot Analysis of MX2 Protein

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Cells collected on the day of HIV infection were lysed in RIPA buffer (50 mM Tris pH 8.0, 0.1% SDS, 1% Triton X-, 150 mM NaCl, 1% deoxycholic acid, 2 mM PMSF). Cell extracts were prepared in 4X LDS Buffer (Invitrogen #NP007) and 2X MES SDS Running Buffer (Invitrogen #NP002). After boiling at 100C for 5 min, 20 μg of the prepared samples were loaded into a NuPAGE 4–12% Bis-Tris Gel (Invitrogen #NP0321) and resolved. Following transfer onto a nitrocellulose membrane (Invitrogen #LC2001), blots were blocked in blocking buffer (PBS, 0.1% Tween, 5% milk) for 1 h, incubated with the MX2 primary antibody (Santa Cruz Biotech #sc-271527; 1:100) overnight, probed with secondary antibody (Cytiva #NA931, 1:1000) for 1 h, and visualized by chemiluminescence (Cytiva #RPN2232) on a Bio-Rad ChemiDoc Touch. Membranes were washed in PBST (PBS, 0.1% Tween) in between each step. After imaging the MX2 stain, membranes were stripped (ThemoFisher #46430), washed, blocked, probed with GAPDH:HRP (Bio-Rad #MCA4739P, 1:1000) for 1 h, and imaged with chemiluminescence.

Garaizar J., López-Molina N., Laconcha I., Lau Baggesen D., Rementeria A., Vivanco A., Audicana A, & Perales I. (2000). Suitability of PCR Fingerprinting, Infrequent-Restriction-Site PCR, and Pulsed-Field Gel Electrophoresis, Combined with Computerized Gel Analysis, in Library Typing of Salmonella enterica Serovar Enteritidis. Applied and Environmental Microbiology, 66(12), 5273-5281.

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Western Blot Procedure for Protein Detection

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Cell lysates in 1× Laemmli sample buffer (10 mM Tris pH 6.8; 8% glycerol; 100 mM DTT; 2% SDS) were run on 4–12% Bis-Tris NuPAGE gels (Thermo Fisher Scientific NP0322) in 50 mM Tris; 50 mM MES; 0.1% SDS and blotted onto nitrocellulose membranes for 4 h at 4 °C in 25 mM Tris, 200 mM glycine, 25% (w/w) methanol. After blocking for 1 h at 23 °C with 5% milk in TBST (10 mM Tris pH 8; 150 mM NaCl, 0.05% Tween 20), membranes were incubated with primary antibodies diluted 1:1000 for 4–16 h at 4 °C, washed three times for at least 15 min in TBST, incubated with secondary HRP-conjugated antibodies diluted 1:1000 in 10% goat serum for 1 h at 23 °C and after 3 additional washes in TBST, developed using a chemiluminescent reagent (GE Healthcare RPN2232) in a Gel Doc XR + imager (Biorad 1708195). Uncropped blots and unprocessed scans are provided in the Source Data file.

Paul D., Stern O., Vallis Y., Dhillon J., Buchanan A, & McMahon H. (2023). Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis. Nature Communications, 14, 947.

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Western Blot Analysis of HPV and Cell Signaling

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For Western blot analysis, cells were lysed using lysis buffer (10 mM tris-HCL pH 7.5, 50 mM KCl, 2 mM MgCl₂, 1% Triton X-100, 1 mM DTT, 1 mM PMSF and protease inhibitor cocktail (Roche)). Samples were incubated for 20 min on ice with intermittent mixing and were then centrifuged for 10 min. Protein samples were mixed with 4x Laemmli-buffer-containing β-mercaptoethanol and then heated at 95 °C for 5 min. The samples were separated by SDS-PAGE and subsequently transferred to a PVDF membrane by semi-dry transfer. Membranes were blocked and then incubated overnight with primary antibodies (HPV16 E6 E6-6F4 (Euromedex), HPV16 E7 NM2 (in-house produced), Ras (G12V mutant-specific) D2H12 (Cell signaling technologies), CDKN2A/p16^INK4a EPR20418 (abcam) or β-actin AC-74 (Sigma-Aldrich)). HRP-coupled secondary antibodies (IgG anti-Mouse IgG (H+L)-HRPO (Dianova), IgG anti-rabbit IgG (H&L)-HRPO (Rockland Immunochemicals)) were then incubated with the membrane for 1 h. Chemiluminescence signal was detected after addition of a chromogenic substrate (VWR International # RPN2232) and detected in a Biorad Chemiluminescence Detector (Biorad #ChemiDoc).

Schifflers C., Zottnick S., Förster J.D., Kruse S., Yang R., Wiethoff H., Bozza M., Hoppe-Seyler K., Heikenwälder M., Harbottle R.P., Michiels C, & Riemer A.B. (2023). Development of an Orthotopic HPV16-Dependent Base of Tongue Tumor Model in MHC-Humanized Mice. Pathogens, 12(2), 188.

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