Visualization of molecular weight standards after electroblotting: Detection by means of corresponding antibodies
✍ Scribed by Ole J. Bjerrum; Dr. Frank R. Hinnerfeldt
- Publisher
- John Wiley and Sons
- Year
- 1987
- Tongue
- English
- Weight
- 678 KB
- Volume
- 8
- Category
- Article
- ISSN
- 0173-0835
No coin nor oath required. For personal study only.
✦ Synopsis
The techniques available for detection of molecular weight standards after blotting are surveyed and evaluated. An identical immunodetection procedure for the antigens as well as for the molecular weight markers was evaluated by immunizing rabbits with the whole protein mixture of some commercially available calibration kits for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies against both high and low molecular weight proteins were successfully raised albeit one rabbit failed to generate antibodies against ovalbumin. For immunodetection of rabbit myosin it was necessary to supplement the immunogen with bovine myosin. Best results were obtained by using more than one animal immunized with kits of different origin. Using alkaline phosphatase conjugated secondary antibodies, as little as 0.6 and 1.2 1. 18 of the total low and high molecular weight mixture, respectively, are necessary for the immunoenzymatic detection of all bands. In fused rocket immunoelectrophoresis such antibodies are also useful for determination of the elution position of the molecular weight markers in gel filtration experiments.