Western Blot Analysis of Protein Levels

For analysis of protein levels by Western blot, whole tissue lysates were used from either the proteomic analysis or were prepared as described previously (34 (link)). Proteins were separated on precast SDS-PAGE 4–12% criterion gels (Bio-Rad) and transferred to polyvinylidene difluoride or nitrocellulose membranes. Site-specific antibodies directed to acetylated α-tubulin (Sigma, T7451), HSPA1 (Enzo Life Sciences, ADI-SPA-810), HSPA2 (Proteintech group, 66291-1), HSPB1 (Enzo Life Sciences, ADI-SPA-800), HSPB5 (Enzo Life Sciences, ADI-SPA-223), HSPB7 (abcam, ab150390), HSPD1 (Enzo Life Sciences, ADI-SPA-805), HSPA4 (Cell Signaling, 3303S), HSP90 (Cell Signaling, 4874S), α-tubulin (Sigma, T9026), tyrosinated tubulin (Sigma, T9028), detyrosinated tubulin (abcam, ab48389), and GAPDH (Cell Signaling, 2118S; Fitzgerald, 10R-G109a) were used to detect the proteins which were visualized with an enhanced chemiluminescence detection kit (Amersham) and scanned with Amersham Imager 600. Protein levels were determined by densitometric analysis and normalized to GAPDH.

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Schuldt M., Dorsch L.M., Knol J.C., Pham T.V., Schelfhorst T., Piersma S.R., dos Remedios C., Michels M., Jimenez C.R., Kuster D.W, & van der Velden J. (2021). Sex-Related Differences in Protein Expression in Sarcomere Mutation-Positive Hypertrophic Cardiomyopathy. Frontiers in Cardiovascular Medicine, 8, 612215.

Publication 2021

SDS-PAGE 4–12% criterion gels α-tubulin HSPA2 HSPB7 HSPD1 HSPA4 HSP90 GAPDH enhanced chemiluminescence detection kit

Antibodies Chemiluminescence Densitometric Gapdh Gels Hsp90 Hspb1 Hspb5 Membranes Nitrocellulose Polyvinylidene difluoride Protein Sds page Tissue Tubulin Western blot α tubulin

Corresponding Organization : Amsterdam University Medical Centers

Other organizations : Victor Chang Cardiac Research Institute, Robert Bosch (Australia), University of Sydney, Erasmus University Rotterdam

Top 5 similar protocols

Variable analysis

independent variables

Protein levels

dependent variables

Acetylated α-tubulin
HSPA1
HSPA2
HSPB1
HSPB5
HSPB7
HSPD1
HSPA4
HSP90
α-tubulin
Tyrosinated tubulin
Detyrosinated tubulin
GAPDH

control variables

Whole tissue lysates from proteomic analysis or prepared as described previously (34)
Proteins separated on precast SDS-PAGE 4–12% criterion gels (Bio-Rad)
Proteins transferred to polyvinylidene difluoride or nitrocellulose membranes
Site-specific antibodies used to detect proteins
Proteins visualized with enhanced chemiluminescence detection kit (Amersham)
Protein levels determined by densitometric analysis and normalized to GAPDH

positive controls

No positive controls specified

negative controls

No negative controls specified

Annotations

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