The primary antibodies were optimized by use of a serial dilution of mixed human muscle standard lysate to ensure that the band signal intensities were located on the linear part of the antibody‐specific standard curve. Primary antibodies targeting the investigated Na+/K+ pump subunits and phospholemman (FXYD1) were monoclonal Na+/K+ pump α1 (alfa6F, Developmental Study Hybridoma Bank, University of Iowa, United states of America), polyclonal α2 (07‐674, Millipore), monoclonal β1 (MA3‐930, Thermo Scientific), and polyclonal FXYD1 (13721‐1‐AP, Datasheet). Polyclonal MCT4 (AB3316P, Millipore) and monoclonal NHE1 (MAB3140, Chemicon) were used to detect the expression of MCT4 and NHE1. Applied antibodies targeting antioxidative and glycogen‐level‐related proteins were polyclonal SOD1 (574597, Millipore) and polyclonal SOD2 (06‐984, Millipore) (Kindly provided by Prof. H. Pilegaard, University of Copenhagen), polyclonal KAT (ab1877, Abcam), polyclonal GLUT4 (PA1‐1065, Thermo Fisher Scientific), and polyclonal GS (3893, Cell Signaling Technology). The secondary antibodies used were HRP‐conjugated goat anti‐rabbit (4010‐05, Southern Biotech), rabbit anti‐sheep (P‐0163, DAKO), and goat anti‐mouse (P‐0447, DAKO). A representative example of Western blotting is provided in Fig. 2 .
P0163
Manufactured by Agilent Technologies
The P0163 is a laboratory instrument designed for performing various analytical tasks. It features a compact and durable construction, enabling reliable operation in laboratory environments. The core function of the P0163 is to provide precise measurements and data analysis capabilities to support scientific research and testing activities.
Automatically generated - may contain errors
Lab products found in correlation
Af2859, by R&D Systems (2 mentions)
P0447, by Agilent Technologies (1 mentions)
Hrp conjugated goat anti rabbit, by Southern Biotech (1 mentions)
Ab3316p, by Merck Group (1 mentions)
Pa1 1065, by Thermo Fisher Scientific (1 mentions)
Ab1877, by Abcam (1 mentions)
Complete mini, by Roche (1 mentions)
Haematoxylin, by Thermo Fisher Scientific (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Ecl substrate, by Thermo Fisher Scientific (1 mentions)
A3853, by Merck Group (1 mentions)
4 protocols using p0163
1
Optimization of Na+/K+ Pump Antibodies
2
Immunoblot Analysis of Mitochondrial and Lipogenic Proteins
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SAT samples were homogenized in extraction buffer [10 mM HEPES, pH 7.5/5 mM EDTA/5 mM dithiothreitol/5 mM MgCl2/PI (Complete Mini, Roche)]. Protein was analysed by immunoblot as previously described,25 (link) using antibodies against cytochrome c oxidase subunit 2 and 4 (COX2 and COX4) (A-6404 and A-21347, Invitrogen); PPARG and sterol regulatory element binding transcription factor 1 (SREBP1) (Sc-1984X and Sc-367X, respectively, Santa Cruz Biotechnology); and β2-microglobulin (B2M) (P0163, Dako Cytomation). Chemiluminescence was developed using horseradish peroxidase-conjugated secondary antibodies (170-6510, Bio-Rad and 711-135-152, Jackson Immunoresearch) and Immobilon ECL Plus kit (Millipore). ODs were quantified using the Multigauge 3.0 software suite (Fujifilm) and normalized to total protein content.
3
Immunohistochemical Analysis of Siglec-6 Expression
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Immunohistochemistry analysis was performed as previously described [7] (link). Briefly, 8 m formaldehyde fixed paraffin embedded (FFPE) tissue sections from NP were rehydrated and blocked in 10 % fetal calf serum (FCS) in PBS supplemented with 1 % 0.01M Tween-20 (PBS-T) for 1 hour. Tissue sections were incubated with polyclonal sheep anti-Siglec-6 (2 µg/ml in 1 % FCS in PBS-T, AF2859, R&D systems) at 4 °C overnight. Sections incubated with 1 % FCS with no antibody served as the negative control. Sections were then incubated with HRP-conjugated secondary antibody anti-sheep (1/200 in 10 % FCS in PBS-T, P0163, Dako Cytomation) for 1 hour at room temperature prior to visualisation with DAB (Vector laboratories) and counterstaining with haematoxylin (ThermoFisher).
4
Western Blot Analysis of Siglec-6 in PL and STBEV
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Western blots were run under reducing conditions with the same protein mass (20 µg, normalised based on bicinchoninic acid assay results) of either PL or STBEV samples.
Membranes were blocked for 1 h with 5 % Blotto (2BScientific) in 0.1 % TBS-T before staining overnight with polyclonal Siglec-6 sheep (0.8 µg/ml, AF2859, R&D systems) or loading control Actin antibody (0.5 µg/ml, A3853, Sigma Aldrich). Membranes were subsequently incubated with HRP-linked secondary antibodies for 1h: anti-mouse (1/5,000, Cell Signalling Technology Inc.) and anti-sheep (1/4,000, P0163, Dako Cytomation) before treatment with enhanced chemiluminescence (ECL) substrates (Thermo Scientific) and exposure. PL immunoblot normalisation of protein expression was done against loading control and band intensities were calculated using ImageJ while for STBEV, we loaded the same mass of total protein.
Membranes were blocked for 1 h with 5 % Blotto (2BScientific) in 0.1 % TBS-T before staining overnight with polyclonal Siglec-6 sheep (0.8 µg/ml, AF2859, R&D systems) or loading control Actin antibody (0.5 µg/ml, A3853, Sigma Aldrich). Membranes were subsequently incubated with HRP-linked secondary antibodies for 1h: anti-mouse (1/5,000, Cell Signalling Technology Inc.) and anti-sheep (1/4,000, P0163, Dako Cytomation) before treatment with enhanced chemiluminescence (ECL) substrates (Thermo Scientific) and exposure. PL immunoblot normalisation of protein expression was done against loading control and band intensities were calculated using ImageJ while for STBEV, we loaded the same mass of total protein.
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