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Published on: September 26, 2020
Multiple antigen detection (MAD) western blotting
Stan Krajewski1, Xianshu Huang, Maryla Krajewska
1Burnham Institute for Medical Research, 10901 N. Torrey Pines Rd, La Jolla, CA, 92037, USA. stan@burnham.org
Methods in Molecular Biology (Clifton, N.J.)
|April 21, 2009
Summary
Sequential detection of multiple antigens (MAD) on a single blot streamlines protein analysis without antibody stripping. This method preserves protein integrity, enabling over 12 antibody reprobings with strong signals for efficient comparative studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunotechnology
Background:
- Traditional immunoblotting requires antibody stripping between detection steps, leading to protein loss and reduced signal.
- Sequential detection of multiple proteins on a single blot is challenging due to antibody interference and background noise.
Purpose of the Study:
- To develop a novel method for sequential detection of multiple antigens (MAD) on a single protein blot without antibody stripping.
- To optimize a horseradish peroxidase (HRPase)-based immunodetection protocol for enhanced signal and reduced background.
Main Methods:
- A modified ECL immunodetection method allowing sequential probing of the same membrane with different primary antibodies.
- Utilized both chemiluminescent and colorimetric substrates for HRPase-based detection.
- Implemented a pre-probing colorimetric development step after secondary antibody incubation to reduce background.
Main Results:
- Enabled detection of >= 12 different primary antibodies on a single blot without significant protein loss.
- Maintained strong signal intensities across multiple sequential antibody probings.
- Demonstrated significantly reduced background noise in ECL detection.
Conclusions:
- MAD immunoblotting offers a streamlined approach for comparative protein expression analysis from limited biological samples.
- The technique preserves protein integrity and signal strength, making it valuable for rare cell populations and clinical specimens.
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01:15Western Blotting
Western blotting is an analytical technique for protein identification. It has various applications in immunology and medicine, including detecting diseases like bovine spongiform encephalopathy, mad cow disease, and human and feline immunodeficiency virus from biological samples.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
02:57Southern Blot
Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...