Immediately following dissection, Gastroc and Quad muscles from WT mice were minced with a sterile scalpel, placed in 5 mL Nonidet P-40 (NP-40) lysis buffer, 1% (w/v) NP-40 (Millipore-Sigma, St. Louis, MO, USA) in 50 mM Tris buffer with 150 mM NaCl, 1 mM EDTA (Invitrogen, Grand Island, NY, USA), and cOmplete EDTA-free Protease Inhibitor Cocktail (Millipore-Sigma) and rocked overnight at 4°C. Supernatant was transferred to 200 μL Wheat Germ Agglutinin (WGA)-agarose (EY Laboratories, San Mateo, CA, USA) and rocked overnight at 4°C. Precipitated sample was washed thoroughly in 0.1% (w/v) NP-40 in 50 mM Tris buffer with 150 mM NaCl, then eluted in 500 μL 0.3 M N-acetyl-D-glucosamine (GlcNAc; Millipore-Sigma, USA) in 0.1% NP-40 buffer for 20 min with rocking at 4°C. To reduce GlcNAc concentration, we dialyzed eluted samples in 1,000-fold volume 0.1% NP-40 buffer overnight at 4°C using 10K MWCO Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher Scientific).
10k mwco slide a lyzer dialysis cassettes
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 10K MWCO Slide-A-Lyzer Dialysis Cassettes are designed for dialysis of protein samples. The cassettes have a molecular weight cut-off (MWCO) of 10,000 Daltons, allowing the removal of small molecules while retaining larger proteins.
Automatically generated - may contain errors
Lab products found in correlation
Microfluidic mixer, by Precision Nanosystems (2 mentions)
Np 40, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Np 40, by Merck Group (1 mentions)
Nonidet p 40, by Merck Group (1 mentions)
Freedom evo 200, by Tecan (1 mentions)
Mabselect sure lx, by GE Healthcare (1 mentions)
Optima l 70 xp, by Beckman Coulter (1 mentions)
6 protocols using 10k mwco slide a lyzer dialysis cassettes
1
Glycoprotein Enrichment from Muscle Tissue
2
Antibody Purification Using MabSelect SuRe LX
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The clarified cell culture supernatants were loaded onto a Tecan Freedom EVO 200 (Tecan Life Sciences, Männedorf, Switzerland) for antibody purification utilizing miniature columns manufactured by Repligen (Waltham, MA, USA) and packed with MabSelect™ SuRe™ LX (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The antibodies were eluted with 20 mM sodium acetate at pH 3.5 and immediately neutralized with 0.33 M Tris, 1 M sodium acetate pH 8.0 and buffer exchanged into 20 mM sodium acetate pH 5.5 using 10K MWCO Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific, Waltham, MA, USA).
3
Heparin-Agarose Precipitation for Laminin Isolation
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200 μL Heparin-Agarose Type I saline suspension (Millipore-Sigma) was added per milliliter of conditioned media, cell lysate, or muscle lysate to be precipitated and mixed overnight at 4°C. Precipitated samples were washed thoroughly with PBS. For western blotting, precipitated samples were resuspended in 50 μL 1X NuPage LDS Sample Buffer with 100 mM β-mercaptoethanol. 20 μL of the resuspension was boiled and used for immunoblotting. For isolation of micro-laminin proteins, precipitated samples were eluted with a high-salt solution, 200 μL 1.5 M NaCl in 0.01 M Tris-HCl (pH 7.5). To reduce salt concentration, we dialyzed eluted samples in 1,000-fold volume 0.01 M Tris-HCl overnight at 4°C using 10K MWCO Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher Scientific).
4
Lipid Nanoparticle Aptamer Encapsulation
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The LNP-aptaDiRs were prepared in the delivery unit at the HIRM Precision RNA Medicine Core. Briefly, an aqueous phase and an organic phase containing ethanol and fluorescent labelled lipid nanoparticles were combined using a microfluidic mixer (Precision Nanosystems, Vancouver, Canada). After preparation, LNP formulations were dialyzed into 1× phosphate buffered saline (PBS) (pH 7.4) for 2 h in 10 K MWCO Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific, Waltham, MA, USA). The size and zeta potential of LNP formulations was characterized. aptaDiR encapsulation efficiency was evaluated by low range Quanti-iT RiboGreen RNA as previously described (precisionnanosystems.com).
5
Density Gradient Centrifugation for Virus Particle Analysis
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For comparison of particle density between SVA and FMDV by cesium chloride (CsCl) density gradient centrifugation, FMDV A/SAU/26/95 (kindly provided by APHIS FADDL) was cultured in passage 3 BHK-21 cells and inactivated using binary ethylenimine using standard methods. Inactivated virus was PEG precipitated using the same methodology as described in Section 2.2 .
SVA-LP8 and FMDV A/SAU/26/95 PEG precipitated viruses was separately layered on 2 mL cesium chloride 2-step discontinuous gradients, 1.38 g/cm3 over 1.42 g/cm3, prepared in TEN buffer (0.05 M Tris, 0.001 M EDTA, 0.15 M NaCl, pH 7.4). Gradients were centrifuged at 217,485× g for 18 h using a SW40Ti rotor in an Optima L-70 XP ultracentrifuge (Beckman Coulter, Indianapolis, IN, USA). Visible bands were collected and dialyzed against PBS at 4 °C using 10K MWCO Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher Scientific, Waltham, MA, USA) prior to Western blotting, as described inSection 2.4 .
SVA-LP8 and FMDV A/SAU/26/95 PEG precipitated viruses was separately layered on 2 mL cesium chloride 2-step discontinuous gradients, 1.38 g/cm3 over 1.42 g/cm3, prepared in TEN buffer (0.05 M Tris, 0.001 M EDTA, 0.15 M NaCl, pH 7.4). Gradients were centrifuged at 217,485× g for 18 h using a SW40Ti rotor in an Optima L-70 XP ultracentrifuge (Beckman Coulter, Indianapolis, IN, USA). Visible bands were collected and dialyzed against PBS at 4 °C using 10K MWCO Slide-A-Lyzer Dialysis Cassettes (Thermo Fisher Scientific, Waltham, MA, USA) prior to Western blotting, as described in
6
Lipid Nanoparticle Formulation for mRNA
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Lipid nanoparticles containing EGFP mRNA or FLuc mRNA were prepared by combining an aqueous phase (mRNA diluted in 100 mM sodium citrate buffer, pH 3.0) and an organic phase containing ethanol and lipids according to each formulation (Table S2 ) using a microfluidic mixer (Precision Nanosystems, Vancouver, Canada) [30 (link)]. After preparation, LNP formulations were dialyzed into 1X phosphate buffered saline (PBS) (pH 7.4) for 2 h in 10K MWCO Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific, Waltham, MA, USA).
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