Hb-α-syn complex levels in brain tissue, cell lysates, and RBCs were measured by ELISA using a mouse monoclonal anti-Hb antibody (ab77125; Abcam, Cambridge, MA, USA) and biotinylated mouse monoclonal anti-α-syn (3D5) antibody [40 (link)] for capture and detection, respectively. After completion of the immunoreaction, samples were incubated with 100 μl ExtrAvidin alkaline phosphatase (E-2636; Sigma-Aldrich, St. Louis, MO, USA) diluted 1:20,000 in blocking buffer followed by the enzyme substrate p-nitrophenyl phosphate (N1891; Sigma-Aldrich). The reaction was allowed to proceed for 30 min at room temperature, after which the absorbance was read at 405 nm using a Multiskan MK3 microplate reader (Thermo Scientific, UT, USA).
Ab77125
Manufactured by Abcam
Sourced in United States
Anti-SARS-CoV-2 Spike Protein Antibody (Ab77125) is a recombinant antibody that binds to the SARS-CoV-2 spike protein. The antibody is designed for research use only and its core function is to detect the presence of SARS-CoV-2 spike protein in samples.
Automatically generated - may contain errors
Lab products found in correlation
Multiskan mk3 microplate reader, by Thermo Fisher Scientific (1 mentions)
Extravidin alkaline phosphatase e 2636, by Merck Group (1 mentions)
P nitrophenyl phosphate n1891, by Merck Group (1 mentions)
Ab14734, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by Promega (1 mentions)
Horseradish peroxidase, by Vector Laboratories (1 mentions)
Sc 7210, by Santa Cruz Biotechnology (1 mentions)
Anti mouse igg horseradish peroxidase hrp linked antibody, by Cell Signaling Technology (1 mentions)
Ge10600002, by Cytiva (1 mentions)
Picoepd western reagent, by Elpis Biotech (1 mentions)
3 protocols using ab77125
1
Measuring Hb-α-Syn Complex Levels
2
Western Blotting of Cytosolic, Mitochondrial, and Membrane Proteins
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Western blotting was performed as previously described [41 (link)]. Briefly, protein samples (20 μg protein/lane) were separated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. Blots were separately probed with mouse monoclonal 3D5 anti-α-syn (1: 5000) and anti-Hb (1: 1000; ab77125, Abcam, MA, USA) primary antibodies, as well as rabbit polyclonal anti-actin (1: 1000; sc-7210, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-voltage-dependent anion channel (1: 500; ab14734, Abcam, MA, USA), and rabbit polyclonal anti-calnexin (1: 1000; 2433s, Cell Signaling Technology, Danvers, MA, USA) antibodies as loading controls for cytosolic, mitochondrial, and membrane fractions, respectively. The blots were probed with appropriate secondary antibodies conjugated with horseradish peroxidase (1:5000; Vector Laboratories, Burlingame, CA, USA). Immunoreactivity was detected using enhanced chemiluminescence reagent (Promega, Madison, WI, USA).
3
Protein Extraction and Western Blot Analysis
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3 ul of separation buffer solution was taken from the chamber and treated with 1.5 ul of protein lysis buffer solution (#9806, Cell Signaling). Protein extraction was done following the manufacture’s protocol. Extracted protein samples were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (#456-1096, Bio-Rad). After electrophoresis (200 V, 40 min), transfer (200 V, 200 mA, 40 min) was made to the nitrocellulose (NC) membrane (GE 10600002, Cytiva). NC membrane was blocked with 1% bovine serum albumin (BSA, A7906, Sigma) in 0.1M Tris-buffered saline (TBS, #28358, Thermo Scientific™) at room temperature. After incubation with anti-human hemoglobin antibody (1:1000 in 1% BSA in TBS solution, ab77125, Abcam) for 1 hour at room temperature, washing was done three times with 0.1M TBS for 10 minutes. After incubation with anti-mouse IgG horseradish peroxidase (HRP)-linked antibody (#7074, Cell Signaling) for 1 hour at room temperature, washing was done three times with 0.1M TBS for 10 minutes. NC membrane was reacted with PicoEPD Western reagent (#EBP1073, ELPIS-BIOTECH) for enhanced chemiluminescence (ECL) reaction. ECL image was taken with Gel Doc system (GDS-200, Optimity, China) and band density was measured with NIH ImageJ software.
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