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Protocols have been used by cail.cn

This section is generated from parts of my dissertation, including all the experimental protocols I used in Bear Lab - UNC. If you have questions about these protocols, please feel free to email me. Thanks!

The yellow highlighted part is the correction to the original PDF file.

A.1 General materials and methods

A.2 Coronin 1B antibody production and affinity purification

A.3 Cell culture and viral transduction

A.4 Molecular cloning

A.5 TaqMan qRT-PCR

A.6 Drosophila S2 cell actin cable formation assay

A.7 Recombinant Coronin 1B protein production

A.8 Actin co-sedimentation

A.9 Pyrene actin polymerization assay

A.10 Pyrene actin depolymerization assay

A.11 Actin TIRF assay

A.12 Second-wave actin debranching assay

A.13 Arp2/3 dissociation assay

A.14 Partial proteolysis and circular dichroism analysis

A.15 Immunoprecipitation

A.16 In vitro phosphorylation

A.17 In vivo phosphorylation

A.18 Peptide mapping

A.19 In vivo Coronin 1B dephosphorylation assay

A.20 Light microscopy and image analysis

A.21 Lamellipodial co-localization analysis

A.22 Active/total Cofilin ratio imaging

A.23 Analysis of free actin filament barbed ends in vivo

A.24 Negative stained samples to TEM

A.25 Platinum replica electron microscopy

A.26 PMA-induced ruffling assay

A.27 Single cell tracking

A.28 Kymography analysis

A.29 Actin retrograde flow measurement

B.1 2-D phospho-peptide mapping

B.2 On-grid immuno-gold staining for electron microscopy

B.3 Prepare grid for whole-mount electron microscopy

B.4 Platinum replica EM (from Svitkina and Borisy)

B.5 Combined TIRF assays

B.6 293Q mammalian protein purification system

B.7 Fluorescently label Arp2/3 complex

B.8 Acid wash glass coverslips

B.9 Prepare 4% paraformaldehyde fixation buffer

In step one, for each 800 ml Buffer, add 0.264g MgCl2-6H2O and 0.166g NaH2PO4-H2O

B.10 Immunofluorescent staining of fixed cells

B.11 Live cell staining by CellTracker

B.12 Microscopy media for live cell imaging without CO2 supply

B.13 Transferrin uptake assay

B.14 ATP depletion/recovery assay

B.15 Coronin 1B protein expression and purification from S2 cells

B.16 Produce Coronin 1B protein in E.coli by TVH system

B.17 In vitro Slingshot phosphatase assay

B.18 Triton X-114 phase separation

B.19 Arp2/3 ATP hydrolysis assay

B.20 PKC kinase activity assay

B.21 Chloroform/methanol precipitation

B.22 Polyclonal antibody affinity purification

B.23 Dialysis tubing preparation

B.24 Home-made sliver staining (Googled)

B.25 Prepare electroporation-competent cells and DNA transformation

B.26 Freeze E.coli cells

B.27 Freeze mammalian cells

B.28 Bio-Rad DC protein assay

B.29 Fugene 6 transient transfection

B.30 Using PolyFect transient transfect Rat2 fibroblasts

The dish used for transfection is 60mm x 15mm style (for example: Polystyrene cell culture dish from Corning, 430166). Serum starvation is overnight.

B.31 DSP cross-linking

B.32 Activation of sodium orthovanadate

B.33 General media and solutions

Bacto ™ has a product called Terrific Broth (0438-17-0), which has additional KH2PO4 9.4g and K2HPO4 2.2g for each liter.
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