Cell media samples were concentrated 10 times using Vivaspin 500 columns (Sartorius Stedim Lab Ltd, Stonehouse, UK) for the APOB-100 detection. Cell lysates as well as concentrated and diluted cell media samples were mixed with Laemmli buffer containing 2-mercaptoethanol (ratio sample: Laemmli buffer = 4:1 v/v) and incubated for 5 min at 95 °C. Proteins were size-separated by SDS-page (6% acrylamide gel, 90 V, 3 h at 4 °C for APOB-100 and calnexin detection; 10% acrylamide gel, 130 V, 1 h at room temperature for albumin detection) and transferred onto a nitrocellulose membrane (0.2 A, 2.5 h at 4 °C). Nitrocellulose membranes were incubated overnight with primary antibodies, washed 3 times for 10 min with tris-buffered saline containing 0.2% tween (0.2% TBS-T buffer), incubated 1 h with HRP-conjugated secondary antibodies, washed 3 times for 10 min with 0.2% TBS-T buffer, incubated with Immobilon Western chemiluminescent HRP substrate (Millipore Corporation, Billerica, Massachusetts, USA). Bands were visualized using Chemidoc XRS System (Biorad, Hercules, California, USA). The following antibodies were used: mouse anti-APOB (Santa Cruz Biotechnology, Dallas, Texas, USA), rabbit anti-calnexin (Sigma-Aldrich, Saint Louis, Missouri, USA), mouse anti-albumin (Sigma-Aldrich, Saint Louis, Missouri, USA).
Mouse anti albumin
Manufactured by Merck Group
Sourced in United Kingdom
Mouse anti-Albumin is a laboratory reagent used in various immunological and biochemical analyses. It is a monoclonal antibody that specifically binds to the albumin protein, which is the most abundant protein found in the blood. This product can be utilized as a tool for the detection, quantification, and purification of albumin in various sample types.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xrs system, by Bio-Rad (3 mentions)
Rabbit anti calnexin, by Merck Group (2 mentions)
Chemiluminescent hrp substrate, by Merck Group (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Mouse anti v5, by Thermo Fisher Scientific (2 mentions)
Mouse anti lpl, by Merck Group (2 mentions)
Mouse anti apoa1, by Bio-Rad (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Rabbit anti calnexin, by Abcam (1 mentions)
3 protocols using mouse anti albumin
1
Western Blot Analysis of APOB-100
2
Western Blot Analysis of Lipid Regulators
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Pre-heparin and post-heparin plasma samples were diluted 1:50 in 0.5-M Tris-HCl (pH 7). The diluted plasma samples, cell lysates, and concentrated media samples were mixed with Laemmli buffer and boiled for 5 min at 95 C. Proteins were size-separated by SDS-PAGE (10% acrylamide gel, 200 V, 45 min) and transferred onto a nitrocellulose membrane (400 mA, 60 min). Membranes were incubated overnight with primary antibodies, washed 2 times for 5 minutes with 0.2% tris-buffered saline Tween, incubated 1 hour with HRP-conjugated secondary antibodies and washed 3 times for 10 minutes with 0.2% tris-buffered saline Tween. After 5-minute incubation with chemiluminescent HRP substrate (Millipore Corporation, Billerica, USA), bands were visualized by Chemidoc XRS System (Biorad, Hercules, CA) and Image Lab Software (Biorad).
The following antibodies were used: mouse anti-LPL (Sigma-Aldrich), rabbit anti-HTGL (Hepatic Triglyceride Lipase) (Sigma-Aldrich), mouse anti-APOA1 (AbD Serotec, Oxford, UK), mouse anti-V5 (Invitrogen), rabbit anti-Calnexin (Abcam), and mouse anti-Albumin (Sigma-Aldrich).
The following antibodies were used: mouse anti-LPL (Sigma-Aldrich), rabbit anti-HTGL (Hepatic Triglyceride Lipase) (Sigma-Aldrich), mouse anti-APOA1 (AbD Serotec, Oxford, UK), mouse anti-V5 (Invitrogen), rabbit anti-Calnexin (Abcam), and mouse anti-Albumin (Sigma-Aldrich).
3
Western Blot Protein Analysis
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Post-heparin plasma samples were diluted 1:50 in 0.5-M Tris-HCl (pH 7). The diluted plasma samples, cell lysates, and concentrated media samples were mixed with Laemmli buffer 5X (SDS 10%, Tris HCl 62.5 mM pH 6.8, glycerol 50%, bromophenol blue 0.01% and β-mercaptoethanol 25%) and boiled for 5 min at 95 °C. Proteins were sizeseparated by SDS-PAGE (10% acrylamide gel, SDS 0.1%, 100 V, 90 min, using running buffer containing 0.1% SDS) and transferred onto a nitrocellulose membrane (400 mA, 60 min). Membranes were incubated for 1 h with primary antibodies, washed 2 times for 10 min with 0.2% tris-buffered saline containing 0.2% tween (TBST), incubated 1 h with HRP-conjugated secondary antibodies, then washed 3 times for 10 min with TBST. Membranes were incubated for 5 min with chemiluminescent HRP substrate (Millipore Corporation, Billerica, MA). Bands were visualized by Chemidoc XRS System (Biorad, Hercules, CA) and quantified using Image Lab Software (Biorad). The following antibodies were used: mouse anti-LPL (Sigma-Aldrich) (1:1000), mouse anti-APOA1 (AbD Serotec, Oxford, UK) (1:500), mouse anti-V5 (Invitrogen) (1:5000), rabbit anti-Calnexin (Sigma-Aldrich) (1:2000) , mouse anti-Albumin (Sigma-Aldrich) (1:1000).
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