Microvesicle lysates were prepared as described above. 26 μl of each sample was loaded to a 4–12 % Bis-Tris Gel (NuPAGE, Life Technologies). Novex® Sharp Standard (Life technologies) was used as a standard. The gel was run for 1 h at 175 V in MES buffer (NuPAGE, Life Technologies) followed by transfer for 1 h at 20 V onto a PVDF membrane (Invitrogen). The membrane was blocked with 5 % BSA in TBS with 0.2 % Tween (TBS-T) for 1 h (RT), followed by washes in TBS-T and incubation with Flotillin-1 (mouse monoclonal, 1:500, BD Biosciences) antibody in 0.5 % BSA in TBS-T over night (4 °C). After extensive washes in TBS-T the membrane was incubated with a peroxidase-conjugated goat anti-mouse IgG (1:20 000, Pierce) antibody in 0.5 % BSA in TBS-T for 1 h (RT) and then finally washed again in TBS-T. The enhanced chemiluminescence (ECL) system (GE Healthcare) was used for development.
Novex sharp standard
Manufactured by Thermo Fisher Scientific
The Novex® Sharp Standard is a pre-stained protein standard used for molecular weight estimation in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It provides a visual reference for monitoring separation and estimating the molecular weights of unknown protein samples during electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence ecl system, by GE Healthcare (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Pierce silver stain kit, by Thermo Fisher Scientific (1 mentions)
2 protocols using novex sharp standard
1
Microvesicle Protein Analysis by Western Blot
2
SDS-PAGE Analysis of XTEN Peptides
Check if the same lab product or an alternative is used in the 5 most similar protocols
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SDS–PAGE was performed
using NuPAGE Bis-Tris 4–12% gradient gels, NuPAGE MOPS SDS
running buffer, and Novex Sharp Standard (Life Technologies). The
thiol-containing XTEN precursors (XTEN-1, 2, 3, 4) were reduced with
10 mM DTT prior to analysis. The XTEN peptide conjugates were loaded
directly on the gels without reduction. Separation was performed for
40 min at 200 V, and the gels were stained using SimplyBlue SafeStain
(Life Technologies) or a Pierce Silver Stain Kit (Thermo Scientific).
using NuPAGE Bis-Tris 4–12% gradient gels, NuPAGE MOPS SDS
running buffer, and Novex Sharp Standard (Life Technologies). The
thiol-containing XTEN precursors (XTEN-1, 2, 3, 4) were reduced with
10 mM DTT prior to analysis. The XTEN peptide conjugates were loaded
directly on the gels without reduction. Separation was performed for
40 min at 200 V, and the gels were stained using SimplyBlue SafeStain
(Life Technologies) or a Pierce Silver Stain Kit (Thermo Scientific).
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