Proteins are heated in SDS that denatures the protein. SDS binds the length of the protein making it negatively charged. The protein is then run on a polyacrylamide gel towards the positive pole. After the gel runs it is electroblotted onto a membrane (often PVDF). The immobilized proteins are incubated with the primary antibody that recognizes the protein of interest. Then a secondary antibody (which is conjugated to an enzyme) is used to bind to the primary antibody. A substrate is added to the blot to allow the visualization of the antibody complex.
DAY 1:
Solutions Needed:
Gel Running Solutions (SDS sample solution, Electrophoresis Buffer, etc.)
Transfer Buffer = add 7.25g Tris-base and 33.5g glycine to 2L H2O and pH to 8.0. Then add 600ml methanol and bring to total volume of 3L.
Dilution Buffer = 1x PBS
Blocking Buffer = 0.25ml Tween-20, 5% Carnation Nonfat Dry Milk (25g), 50ml 10x PBS, 450ml H5O. (MAKE FRESH, KEEP REFRIGERATED.) Makes 500ml.
Note: PBS = Phosphate Buffer Saline.
Gel Electrophoresis:
Run 10% acrylamide gel of AP, β-galactosidase, cell lysate, and molecular weight marker according to the Exp 5.1 protocol.
• Use 9μL of ~1mg/ml stock AP and β-galactosidase, 21μL of cell lysate, and 5μL of Sigma Marker.
• Gels need to be loaded symmetrically. Two groups per gel.
Group A should use lanes 1-4, and Group B should lanes 7-10, leaving 5-7 empty.
• Run gels at 120V until dye line is through the stacking gel, then increase to 150V and run until dye line is less than 1/4 inch from the bottom of the gel.
