Method for the sequential immunochemical evaluation on a same biological sample of NCAM (Neural Cell Adhesion Molecule) protein and its PolySialic Acid (PSA) tail, by western blot coupled to chemiluminiscence detection.

Description of the technology

The method of invention consists in the immunochemical evaluation on a same biological sample of polysialylated forms of NCAM, PSA-NCAM, concatenating two western blots (for the consecutive immunodetection of NCAM and PSA, or vice versa), separated by an antibody stripping step. The procedure is completed by a second stripping step to conduct the loading control with a standard protein, and a final protein colorimetric staining of the membrane to check the transfer efficiency, or alternatively to perform a more representative load control from all protein bands of the sample.

Specifications

The method involves the immunochemical assessment on a same biological sample of NCAM and PSA (in this order or vice versa) by two consecutive western blots with specific monoclonal antibodies, coupled to detection by chemiluminiscence. The second western blot is preceded by antibody stripping to allow detection of the second molecule, and is followed by another antibody stripping to perform the protein load control by western blot of a housekeeping protein (GADPH, actin, etc). Finally, the staining of membrane proteins by a conventional method (Coomassie brillant blue, etc) it is recommended with the dual purpose of checking transfer efficiency, and to achieve a load control of higher reliability from all proteins of the sample. This method provides higher efficiency to the usual independent immunochemical analysis of NCAM and PSA.

Main advantages of its use

• Quantitatively, the method decreases by 50% the consumption of biological sample and several other reagents (such as buffers), and allows a considerable saving of time.
• Also, a single analysis allows to know the protein expression and its polysialylation status, an information about the NCAM structure which is essential for understanding the biological activity of this protein.
• From a qualitative point of view, the designed method reduces the possibility of experimental error by incorporating the valoration of NCAM and PSA in two coupled steps of the same assay, so that both, the physicochemical conditions and operative handling of the analysis, remain more easily controlled

Applications

• The procedure falls within the field of molecular biology. Its potential applications are related to basic research of PSA-NCAM isoforms (their biological structure and function), as well as to applied research studies (clinical trials), that are concerned with the evolution in patients of NCAM expression and its degree of polysialylation. The profile of its potential users corresponds to basic molecular biologists and clinical researchers who need to carry out analysis of PSA-NCAM expression in small or restricted (human) samples.