Approaches that can be used to study expression of specific proteins

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1 Approaches that can be used to study expression of specific proteins Receptors and transporters Homogenate binding studies Receptor autoradiography Radiochemical Western blotting Immunohistochemistry/cytochemistry Immunochemical

2 Approaches that can be used to study expression of specific proteins Enzymes Immunhistochemical/cytochemical analysis/immunoblotting for the presence of enzyme molecules Enzyme assay functional activity

3 Approaches that can be used to study expression of specific proteins Transcription factors Expression using Western blotting Expression using immunohistochemistry Phosphorylation state (CREB vs pcreb) Western blotting/elisa DNA binding (EMSA assay)/elisa based DNA binding assays Determine cytosolic vs. nuclear location?

4 Measurement of protein in brain tissue Bradford assay dye binding assay Measure protein in: - Tissue homogenate Analyse data Generic Skills: - Pipetting skills - Serial dilutions - Data analysis Equalize tissue for protein concentation

5 Western immunoblotting for a specific protein of interest

6 Southern Northern DNA RNA Transcription Splicing Mature mrna Nucleus Cytoplasm Translation Western Proteins

7 7 Steps- Western/Protein Blotting Tissue Preperation Protein Determination SDS-polyacrylamide gel electrophoresis Blotting Labeling Detection Quantification

8 Tissue Preparation/Extraction of protein Homogenisation Homogenization Buffer: Protease inhibitors prevent the breakdown of the polypeptide chain by proteases Phosphatase inhibitors inhibit protein dephosphorylation DDT, EDTA breakdown various bonds Sucrose gives weight to sample Centrifugation Protein determination v. important: comparisons are made as per total amount of protein present per well

9 Tissue preparation (ctd) Sample added 1:1 with sample buffer Sample buffer contains: SDS: Breaks down quaternary, tertiary, and secondary structures of proteins. β-mercaptoethanol breaks the disulphide bonds in multisubunit proteins. Bromophenol Blue Dye: allows monitoring of electrophoretic run. Glycerol: gives sample weight

10 Electrophoresis Study of the movement of charged molecules in an electric field (Faraday 1833: passage of electricity through solution). Uses: Analysis and purification of biomolecules especially proteins, nucleic acids, peptides and nucleotides. May also be used to determine molecular weights Particles migrate through a solvent that is supported be a homogenous medium (eg) gel. Gel acts as a sieve. Gel types: Polyacrylamide/agarose/agarose-acrylamide Polyacryamide molecular sieving can be controlled by the concentration of the gel and the absorption of proteins is minimal.

11 Polyacrylamide Gels Are formed from the polymerisation of acrylamide monomer is the presence of smaller amounts of N,N - methylene-bis acrylamide (bis-acrylamide) bis-acrylamide=crosslinking agent. Polymerised in a head to tail fashion which is initialised by ammonium persulfate (APS) and TEMED.

12 SDS-PAGE (Maizel, 1966) At a neutral ph, in 1% SDS and 0.1 M mercaptoethanol (within gel), most multichain proteins bind SDS and dissociate. Complexes consisting of protein subunits and SDS behave as though they have uniform shape and an identical charge-to-mass ratio.

13 SDS-PAGE (ctd) Overall charge is negative due to binding of SDS Proteins migrate towards the anode due to negative charge. The proteins then separate according to size with the gel acting as a molecular sieve. Pore size of the gel is variable (% acrylamide) Low percentage i.e. high pore size is required for high molecular weight proteins and vice versa

14 SDS PAGE Denaturing electrophoresis -Multisubunit proteins give multiple bands on a gel

15 Blotting Pore size of the gel limits penetration by antibodies Blotting onto a porous membrane can be achieved giving a mirror image of the gel. Nitrocellulose: versatile, easy to use, no activation required, brittle when dry PVDF: no blow through of proteins, good mechanical strength, requires immersion in methanol

16 Labeling & Detection Light Hyper film-ecl

17 Densitometry converts band density into numbers Densitometry Sample immunoblot

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