Laemmli buffer containing 4% SDS, 10% β-mercaptoethanol (M6250; Sigma-Aldrich), 20% glycerol (GI345; Melford), 0.004% blue bromophenol (A2331,0025; AppliChem), and 0.125 M Tris-HCl was added to protein lysates, followed by boiling at 95°C for 10 min. Samples were separated on SDS-PAGE and transferred to a nitrocellulose (Macherey-Nagel) or a polyvinylidene difluoride (Merck) membrane. The membranes were blocked with 5% milk (A0830; PanReac AppliChem) and probed with the following antibodies: anti–β-Actin (ab8227; Abcam), anti–β-Tubulin (ab15568; Abcam), anti-CBS (14787-1-AP; Proteintech), anti-CSE (12217-1-AP; Proteintech), anti-MPST (HPA001240; Atlas Antibodies), anti-MTCO1 (ab14705; Abcam), anti-Citrate Synthase (ab96600; Abcam), anti-SOD2 (13141; Cell Signaling), anti-TOM40L (ab236421; Abcam), anti-TIM50 (ab109436; Abcam), anti-HIF1α (sc-13515; Santa Cruz Biotechnology), anti-H3 (ab176842; Abcam), anti-V5 (Merck), anti-HA (H6908; Sigma-Aldrich), and anti-Biotin (HRP conjugate; 5571; Cell Signaling). Immunoblots were next incubated with a secondary antibody (anti-rabbit [AP132P; Merck] or anti-mouse [7076; Cell Signaling]) and visualized using the Western HRP substrate (Merck). ImageJ software was used to quantify the expression levels of the proteins.
A0830
Manufactured by ITW Reagents
Sourced in United States
The A0830 is a laboratory-grade piece of equipment. It is designed for precise and accurate measurements within a laboratory setting. The core function of this product is to provide reliable and consistent results for various applications.
Automatically generated - may contain errors
Lab products found in correlation
Tc287, by HiMedia (2 mentions)
Chemidoc xr, by Bio-Rad (2 mentions)
Ab14705, by Abcam (1 mentions)
Ab96600, by Abcam (1 mentions)
Polyvinylidene difluoride, by Merck Group (1 mentions)
Ab109436, by Abcam (1 mentions)
Ab176842, by Abcam (1 mentions)
M6250, by Merck Group (1 mentions)
Sc 13515, by Santa Cruz Biotechnology (1 mentions)
Ab15568, by Abcam (1 mentions)
H6908, by Merck Group (1 mentions)
Ap132p, by Merck Group (1 mentions)
Western hrp substrate, by Merck Group (1 mentions)
Ab8227, by Abcam (1 mentions)
Laemmli buffer 2x, by Merck Group (1 mentions)
Emp011005, by Euroclone (1 mentions)
Hrp conjugated goat anti rabbit, by Cell Signaling Technology (1 mentions)
E ir r301, by Elabscience (1 mentions)
Mini trans blot, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
4 protocols using a0830
1
Western Blot Analysis of Mitochondrial Proteins
2
Protein Detection via Western Blot
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Detection of specific proteins among the experimental series required a proper sample preparation, in which, for each specimen, the Laemmli buffer 2X (S3401; Sigma-Aldrich) was mixed with an equal protein amount before heat denaturation (5 min at 95 °C). Subsequently, loading a protein range from 15 to 30 µg, electrophoresis was performed in sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). After moving proteins to the nitrocellulose membrane (Amersham Protran Premium; Cytiva, Marlborough, MA, United Stetes), foils were firstly blocked for one hour in no-fat milk (5% w/v) (A0830; PanReac Applichem, Chicago, IL, USA) and then incubated overnight at 4 °C with specific primary antibodies. The next morning, HRP secondary antibodies, recognizing the related primary species, were applied to the membrane for 1 h at room temperature. Finally, chemiluminescence was detected with the Chemi Doc XRS (Bio-Rad, Hercules, CA, USA) instrument using liteablot chemiluminescent as a substrate (EMP011005; EuroClone). Each incubation step was preceded and followed by three washes in TBS plus 0.05% Tween-20 (TC287; HIMEDIA, Mumbai, India) (T-TBS).
3
Western Blot Analysis of PKA-Cα
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Approximately 10–30 µg of cell protein extract and 5–50 µg of serum or urine proteins were loaded in 10% acrylamide gels. Later, proteins were transferred onto a nitrocellulose membrane (GEH10600008, Cytiva, Marlborough, MA, USA) by Mini Trans-Blot (Bio-Rad Laboratories, Hercules, CA, USA). In order to reduce both noise and aspecific bond, potential free spots on nitrocellulose film were covered with non-fat milk (5% w/v) (A0830, PanReac Applichem, Chicago, IL, USA) for one hour. Anti-PKA-Cα (sc-903, Santa Cruz Biotechnology, Dallas, TX, USA) was subsequently incubated overnight at 4 degrees. The next day, HRP-conjugated goat anti-rabbit (#7074, Cell Signaling Technology) was added to the membranes for one hour at room temperature. Washing with TBS Tween-20 (TC287, HIMEDIA, Mumbai, India) preceded and followed each single procedure. Lastly, using enhanced chemiluminescence reagent (E-IR-R301, Elabscience, Houston, TX, USA), immunoblotting bands were detected and acquired by Chemi-Doc XRS (Bio-Rad Laboratories).
4
Western Blot Analysis of Protein Expression
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Cells were harvested by centrifugation and resuspended in lysis buffer (NaCl 150 mM, Tris HCl pH 8 50 mM, EDTA 2 mM, NP-40 0.5%, glycerol 10%, protease inhibitor cocktail (Sigma Aldrich#P8340). A total of 40 ug of total protein extract were loaded on a 12% SDS PAGE gel and gels were blotted by semi-dry protein transfer to 0,45 µm nitrocellulose membrane (GE10600002 Amersham Protran). After membrane blocking with 5% (v/v) non-fat milk (A0830 PanReac Applichem) in TBS-tween 0.1%, membranes were incubated with specific antibodies and developed with chemiluminescence reagents (Supersignal West Pico PLUS Chemiluminescent Substrate Thermo Scientific (Waltham, MA, USA) 34577 for GAPDH and β-tubulin Western blot; Immobilon Western Merck WBKLS0500 for FOS Western blot). Images were acquired with Chemidoc MP Imaging System Bio-rad.
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