Immunoblotting is primarily used for protein separation and identification. The brief steps are, isolating proteins by gel electrophoresis, transferring proteins to a membrane, using polyclonal or monoclonal antibodies to identify proteins by fluorescence or chemiluminescence detection.
Here we go over the immunoblotting protocols, including the reagents, solutions, procedures, and troubleshooting advice for typical issues. Our aim is to help you understand and explain the protocol processes and take full advantage of the detailed protocols for immunoblotting.
|Stages||Solutions and Reagents|
|Cell Lysis||Phosphate buffer saline (PBS), cell lysis buffer, protease inhibitors|
|SDS-PAGE||Stacking gel buffer, separating gel buffer, electrophoresis buffer, loading buffer, running buffer|
|Gel Transfer||Transfer buffer, blocking buffer|
|Immunoblotting||Primary antibody, secondary antibody, antibody dilution buffer, membrane washing buffer|
Workflow of Immunoblotting
Proteins can be extracted from different sample types like cells and tissues that will go through ice-based procedures including cell washing, digestion, transfer, agitation, and centrifugation. Tissue samples should firstly be dissected, homogenized and lysed, and agitated, and the remaining steps are essentially the same as for cell samples. Determine the volume of lysate to be used for protein analysis, add sample buffer, and heat to reserve.
The percentage of gel depends on the size of the protein you are interested in, or you can use a gradient gel. Place the gel in the tank and fill with buffer, load the sample in the wells, and run at the appropriate voltage until the dye reaches the bottom of the gel.
Commonly used protein blotting membranes include nitrocellulose and polyvinylidene fluoride. After removing the gel, a transfer sandwich of gel and membrane is created and placed in the device for transfer. The specific running time and voltage should be proportional to the thickness of the gel.
Blocking can prevent non-specific binding of the antibody to the membrane. Place the membrane in the blotting vessel, add enough blocking solution to cover the entire membrane surface, and shake to incubate. This can help to reduce background noise while enhancing the target protein signal.
Primary antibody staining is a critical step. Incubate the membrane with the diluted primary antibodies, wash the membrane at the end of the incubation to remove excess primary antibodies, continue incubation with a good selection of secondary antibodies in the blocking buffer, and finally wash to remove excess secondary antibodies.
Select signal imaging reagents for detection, after which choose the machine of interest for imaging blotting and quantifying the outcomes.
Despite the simplicity of the immunoblotting, problems may still arise such as undetected or weak signals. For some of the problems that may occur, we provide the following troubleshooting guide.
Immunoblotting is a critical technique for protein detection. Creative Biolabs can be the support to secure your ultra-sensitive result. Please browse our immunoblotting protocols and choose from our complementary products and services.
For research use only. Not intended for any clinical use.