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Western Blotting
Western Blotting Protocol (Immunoblotting Protocol)
Western Blotting refers to the electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to sheets of PVDF or nitrocellullose membrane, followed by immunodetection of proteins using antibodies with fluorescent or chemiluminescent detection. a. If using PVDF membrane, wet the membrane in methanol for 15 seconds. Membrane should uniformly Important: To ensure an even transfer, remove air bubbles between layers by aBlot Rolleror carefully rolling a pipette or a stirring rod over the surface of each layer in the stack. Do not apply excessive pressure to prevent damaging the membrane and gel.Western Blotting Workflow Steps
a)Tank Transfer
b)Semi-dry Transfer
a)Traditional Immunodetection
b)30-minute Immunodetection Protocol using the SNAP i.d.®SystemMembrane Transfer (Tank Transfer)Protocol
Materials
Setup
change from opaque to semi-transparent.
b. Carefully place the membrane in Milli-Q®water and soak for 2 minutes.
c. Carefully place the membrane in transfer buffer and let equilibrate for at least 5 minutes.Transfer Stack Assembly
Method for Protein Transfer (Tank Transfer)
Tips for Successful Western Blotting Transfer
IMPORTANT: Do not bump the cathode plate cover during the run since it could disturb the alignment of the transfer stack and cause inaccurate results.Semi-dry Membrane Transfer Protocol
Materials
Setup
a. Wet the membrane in methanol for 15 seconds. The membrane should uniformly change from opaque to semitransparent.
b. Carefully place the membrane in Milli-Q®water and soak for 2 minutes.
c. Carefully place the membrane in anode buffer II and let equilibrate for at least 5 minutes.For single transfers:
For multiple transfers:
For the last gel, go to step 10.Method for Protein Transfer (Semi-dry)
*The surface area (cm2) is calculated from the dimensions of the footprint of the stack on the anode plate. This value is independent of the number of gels in the stack.
*Shaker and Troughs only required for traditional immunodetection; for rapid, vacuum-driven immunodetection using the Antibody Incubations Follow manufacturer’s instructions. The following is a general protocol for fluorescent immunodetection. For optimal results, refer to manufacturer’s protocol provided with the reagents. Note: If using chemifluorescent reagents, follow reagent manufacturer’s directions.Traditional Immunodetection
Materials
-Chemiluminescence substrates
-Chromogenic substrates
SNAP i.d.®system, refer to theSNAP i.d.®immunodetection protocol.
Chromogenic and Chemiluminescent Detection of Proteins
Immunodetection Using BCIP/NBT SubstrateChemiluminescent Detection
Incubate for 1 minute.
Note: A cut-to-size sheet protector or a freezer bag can also be used.Fluorescent Detection
Required Equipment
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