Incubate for 30 min at room temperature with the secondary antibody conjugated to HRP. Incubate for 30 min at room temperature with primary antibody (purified antibody 0.1-10 µg/mL, antiserum 1:1,000-100,000, hybridoma supernatant 1:100-10,000) in BSA/TBS-T. Apply slowly, minimizing the area of solution penetration (usually 2-4 mm in diameter).īlock non-specific sites in 10 cm Petri dishes with 5% BSA in TBS-T by immersion (30 min to 1 h at room temperature). Use a narrow-mouthed pipette tip and spot 2 µL of sample onto the nitrocellulose membrane at the center of the grid. Draw a grid with a pencil to indicate the area where you want to make the blot. TBS-T is a TBS solution containing 0.05% Tween 20.īSA/TBS-T is a TBS-T solution containing 0.1% BSA. TBS is made of 20 mm Tris-HCl and 150 mm NaCl with a pH of 7.5. Creative Biolabs can develop specific antibodies for you to use to perform dot blotting assays. If you have a purified protein and a specific antibody, you can use the dot blotting method to make semi-quantitative estimates of protein concentrations in crude preparations such as culture supernatants. Please keep in mind that Creative Biolabs cannot guarantee specific results for dot blotting assays.ĭot blotting is a technique used to detect, analyze, and identify proteins, which is similar to western blotting, but differs in that protein samples are not separated by electrophoresis, but are spotted directly onto a membrane or paper substrate by means of a circular template. This procedure should be used as a guideline only. With strict regulation and effective execution, we are dedicated to providing the most valuable solutions to complete your projects. Cell-based Membrane Protein Expression and PurificationĬreative Biolabs is offering the most comprehensive services for antibody development projects.Cell-free Membrane Protein Expression and Purification.Membrane Protein Solubilization and Stabilization.Manufacturability Assessment and Optimization.SIAT ® Immunogenicity Assessment Services.CreMap™ Epitope Mapping & Discovery Services.Dual-Polarization Interferometry (DPI) Services.Differential Scanning Calorimetry (DSC) Services.Isothermal Titration Calorimetry (ITC) Services.Magic™ Human Antibody Production Using Transgenic Mice.Bovine Ultralong CDR3 Antibody Humanization.Antibody Off-target Identification and Specificity Profiling Service.Biomolecular Interaction Analysis Services.Epitope Prediction for the Identification of Antibody Epitopes.Epitope Mapping by X-ray Crystallography.Phage Display & Antibody Library Services.

Dot blot