Biology Reference
In-Depth Information
15.4 Protocol 2: Labeling with Fluorescent Probes Using Cyclooctynes .....................276
15.4.1 Step 1: Fluorescent Labeling of Azido-Tagged Rhodopsin
with SpAAC ................................................................................... 277
15.4.1.1 Materials ......................................................................... 277
15.4.1.2 Procedures...................................................................... 277
15.4.2 Step 2: Characterization of Labeled Receptor Using UV-Vis
Spectroscopy and In-Gel Fluorescence ....................................... 278
15.4.2.1 Material ........................................................................... 278
15.4.2.2 UV-Vis Spectroscopy ....................................................... 278
15.4.2.3 In-Gel Fluorescence......................................................... 278
15.4.3 Step 3: Kinetic Study of the SpAAC ........................................... 278
15.4.3.1 Materials for Silver Staining: ............................................. 278
15.4.3.2 Prepare Samples of the SpAAC for time-series Analysis .... 281
15.4.3.3 In-Gel Fluorescence......................................................... 281
15.4.3.4 Silver Staining.................................................................. 281
15.4.3.5 Data Analysis................................................................... 281
15.5 Protocol 3: Preparation of Biotinylated Antibodies ............................................282
15.5.1 Method 1: Biotinylation of Periodate-Oxidized Antibodies with
Biocytin Hydrazide ................................................................... 282
15.5.1.1 Materials ......................................................................... 282
15.5.1.2 Solutions and Buffers....................................................... 283
15.5.1.3 Procedure ....................................................................... 284
15.5.2 Method 2: Biotinylation of Antibodies with Sulfo-NHS-SS-biotin .. 284
15.5.2.1 Materials ......................................................................... 284
15.5.2.2 Solutions and Buffers....................................................... 284
15.5.2.3 Procedure ....................................................................... 285
15.6 Protocol 4: Preparation of Detergent-Solubilized Lipids.....................................285
15.6.1 Materials ................................................................................. 285
15.6.2 Solutions and Buffers ............................................................... 285
15.6.3 Procedure ................................................................................ 286
15.7 Protocol 5: Single-Molecule Immunoprecipitation on Glass-Bottom Microplates .287
15.7.1 Step 1: Cleaning of Glass-Bottom Microtiter Plate ....................... 287
15.7.1.1 Materials ......................................................................... 287
15.7.1.2 Solutions and Buffers....................................................... 287
15.7.1.3 Procedure ....................................................................... 288
15.7.2 Step 2: Functionalize Glass Surface with Epoxy Silane ................ 289
15.7.2.1 Materials ......................................................................... 289
15.7.2.2 Solutions and Buffers....................................................... 289
15.7.2.3 Procedure ....................................................................... 289
15.7.3 Step 3: Immobilization of Biotinylated-Bovine Serum Albumin..... 289
15.7.3.1 Materials ......................................................................... 289
15.7.3.2 Solutions and Buffers....................................................... 290
15.7.3.3 Procedure ....................................................................... 290
