![]() ![]() Kit contents accommodate 32 students (8 4-student workstations) Primary antibody (monoclonal mouse anti-myosin light chain), lyophilized Secondary antibody (polyclonal goat anti-mouse antibody conjugated to myosin peroxidase horseradish (HRP), freeze-dried HRP A color detection reagent HRP B color detection reagent 10x Tris-glycine, 1 L Non-fat dry milk blocker 10x phosphate buffered saline (PBS), 2 x 100 ml Tween 20 10%, 5 ml Nitrocellulose sheets, 0.45 µm (8) Blotting paper sheets (16) Reagent tubes (25) Curriculum including teacher guide, student manual and graphic guide quick. Serves as a lab extension for the Comparative Proteomics I kit: Protein Profiling Duration Hands-on: Lab activity in four sessions, 45 min per session Features and Benefits: Application of Immunology Teaches students to use antibodies as detection tools. In this second part of the lab, students apply western blot techniques to their polyacrylamide gel results to specifically identify the myosin light chain among the hundreds of proteins that make up muscle cell extracts from near and distant fish species. 117179), students use sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to generate protein profiles and visualize the unique array of proteins that make up the muscle tissues of different fish. In the first part of this lab (Comparative Proteomics Kit I: Protein Profiling, ref. Observe the developed film and put another film covering the blotting membrane if needed.The "Comparative Proteomics II: Western Blot Module" kit is the second part of a two-part lab.Develop the film by using a developed machine. Cover the membrane with a chemiluminescence film.Dry the blotting membrane with a filter paper to eliminate excess of ECL.Develop the blotting membrane with 5 ml of ECL for 1 min.Wash the blotting membrane four times with PBS + 0.1% Tween 20 for 15 min each.The proper method for sealing the gel tray with tape is shown along with pouring the molten agarose and storing the gel for later use. Incubate with the corresponding secondary antibody diluted 1:2000 for 1 hour at room temperature. Tutorials Tutorials Casting an Agarose Gel (Duration 2:27 min) This video demonstrates how to cast an agarose gel.Wash the blotting membrane three times with PBS + 0.1% Tween 20 for 15 min each.Use another primary antibody as loading control (tubulin, GAPDH, lamin, etc). Incubate the blotting membrane with the primary antibody for 1 hour at room temperature.Incubate the membrane in PBS + 2.5% dry milk powder overnight at 4º or for two hours at 37º to block non-specific binding to the membrane.(2008) Evaluation of the Criterion Stain Free Gel Imaging System for Use in Western Blotting Applications. Fill the tank with the blotting buffer ( Appendix II) and connect it to the powerPac Power supply 2 hours at 200 mA (or overnight at 20mA). Elbaggari A, Choe J, McDonald K, Alburo A.Put it in the electrophoresis blotting module. Remove the air bubbles by gently rolling a Pasteur pipette over the pad. Prepare the blotting “sandwich” using the gel holding cassette.Cut and wash the blotting membrane in methanol and in blotting buffer for 10 min.Wash the gel in blotting buffer ( Appendix II) 10 min.Connect the tank to the power Pac Power supply and run the gel at 100 V until the gel front reaches the bottom. ![]() Use one well to load a molecular weight standard.Centrifuge the samples and heat for 5 min at 95º, centrifuge again and load into the gel.Fill the tank with the electrophoresis running buffer ( Appendix II).Prepare lysates by mixing with loading buffer and load as follows (in case of transfected cells put between 10-30ug/well, in case of no transfected cells or tissue extracts put 200ug/well).Prepare the stacking gel and put the comb inside it. Prepare the running gel percentage according to the expected molecular weight of the antigen ( Appendix I).ECL detection system (GE Healthcare RPN2109).Conjugated secondary antibody (DAKO polyclonal anti mouse/rat/rabbit HRP).30% acrylamide-bisacrylamide solution (Bio-Rad 161-0158).Nitrocellulose membrane (Millipore IPVH00010).Home > Protocols > Western Blotting (WB) protocol Western Blotting (WB) Protocol created by Lorena Maestre - Monoclonal Antibodies Unit, Centro Nacional de Investigaciones Oncológicas Material ![]()
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