Immunoblot Detection

Abstract

Immunoblotting (often referred to as western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. Following electrophoretic separation of proteins and transfer from the gel to an appropriate membrane, the immobilized proteins are probed with specific antibodies to identify and quantitate any antigens present. After being probed with primary antibody, the membrane is washed and the antibody-antigen complexes are identified using horseradish peroxidase (HRPO) or alkaline phosphatase (AP) enzymes coupled to the secondary anti-immunoglobulin-G (anti-IgG) antibody (e.g., goat anti-rabbit IgG). As described in this unit, the detection enzymes are attached directly or via an avidin-biotin bridge to the secondary antibody. Chromogenic or luminescent substrates are also described for visualizing the activity.

Assays for Acetylation and Other Acylations of Lysine Residues

“…Anti-acetyl Lys (rabbit) antibody against specific residues of target protein (commercially available Abcam and EMD Millipore, or developed in-house using acetyl Lys peptides) Anti-acetyl Lys rabbit polyclonal antibody (e.g., Cell Signaling Technology and ImmuneChem Pharmaceuticals Inc.) Secondary antibody: anti-rabbit IgG, horseradish peroxidase-linked whole antibody (Amersham Biosciences) Chemiluminescent substrate (Supersignal from Pierce, or equivalent) 10-cm culture plates Probe sonicator (e.g., Virsonic from VirTis) End-over-end rotator 0.5 ml tubes, pre-chilled Boiling water bath Nitrocellulose blotting membrane (Pall Life Sciences, BioTrace NT) Platform rocker Additional reagents and equipment for SDS-PAGE (UNIT 10.1; Gallagher, 2012), blot transfer (UNIT 10.7; Goldman et al, 2015), and immunoblot detection (UNIT 10.10; Gallagher, 1996) Express a hyperacetylated FLAG fusion protein in HEK293 cells 1. Seed 4 × 10 5 HEK293 cells per 10-cm plate in 10 to 12 ml of complete DMEM medium on the day prior to transfection.…”

Universal Non‐Antibody Detection of Protein Phosphorylation Using pIMAGO

“…Additional reagents and equipment for SDS-PAGE (Gallagher, 2012) and immunoblotting (Gallagher, 2001, Ursitti et al, 2001.…”

Analysis of Protein Sumoylation

“…Sigma-Aldrich) and 1× complete protease inhibitor (Roche) N-ethylmaleimide (optional) Antibodies to the protein of interest or to the epitope tag of a transfected, tagged protein IgG, species-matched, nonspecific control (Sigma-Aldrich) Protein G-Sepharose or protein A-Sepharose (GE Healthcare) 2× SDS-PAGE sample buffer (see recipe) Blocking buffer: e.g., 5% (w/v) nonfat dry milk Antibodies to SUMO-1, SUMO-2, or SUMO-3 (Thermo Fisher) Enhanced chemiluminescence (ECL) (Gallagher, 2012), electroblotting (Ursitti et al, 2001), and immunoblot detection (Gallagher, 2001) 1. Wash two 100-mm plates of cells with PBS, harvest cells by scraping each plate in 1 ml PBS, transfer to a 2-ml microcentrifuge tube, and collect by centrifuging 30 sec at 13,000 × g, room temperature.…”

“…Analyze samples by SDS-PAGE (Gallagher, 2012) and transfer to a nitrocellulose or nylon membrane (Ursitti et al, 2001 8. Visualize the immunoblot signal using enhanced chemiluminescence (ECL) or enhanced chemifluorescence (ECF) reagents, following the manufacturer's instructions (also see Gallagher, 2001).…”

“…Current Protocols in Protein Science Additional reagents and equipment for SDS-PAGE electrophoresis (Gallagher, 2012), electroblotting (Ursitti et al, 2001), and autoradiography (Gallagher, 2001) 1. Translate the protein to be tested using a commercially available in vitro coupled transcription/translation kit using PCR product or plasmid containing an ORF for the protein of interest and following the kit manufacturer's instructions.…”