Methods and Protocols

Methods and Protocol Collection

This chapter lists all the methods and protocols used in our lab in detail. They are clearly structured and organised according to their respective categories to enable quick orientation and systematic application. The aim is to provide a comprehensive and comprehensible collection of approaches that are relevant to both current work and future references.

Cell Isolation and Cell Culture

• Cultivation of HEK293T cells
• Transfection of HEK293T cells
• Transfection of fibroblasts
• siRNA transfection with INTERFERin
• Isolation of human granulocytes
• Granulocyte FACS phagocytosis assay
• Infection of granulocytes

Production of Viral Particles and Secreted Proteins in Mammalian Cells

• Production of lentiviral particles
• Transduction and titration of lentiviral supernatant
• Viral transduction of mammalian cells via spinfection
• Concentration of lentiviral particles via ultracentrifugation
• Production of soluble CEACAM domains in human cells

Functional Cellular and Biochemical Assays

• Cell viability (MTT Assay)
• Cell Adherence assay
• Determination of Integrin Activity (ELISA-based)
• Protein Phosphatase Assay
• In vitro Tyrosine Kinase Activity Assay
• Covalent coupling of proteins to latex beads
• Fibronectin-Binding Assay
• GST-Pulldown
• Rac pulldown assay (with GST-PBD beads)
• StrepTactin-Pulldown
• Granulocyte oxidative burst
• Luciferase Reporter Assay
• SEAP Reporter Assay
• Measurement of lipid raft localization via flow cytometry
• Preparation of deterg.-resist. membrane microdomains (Sucrose Gradient)
• Measurement of endocytosis via reversible surface biotinylation
• Immunoprecipitation

SDS-PAGE and Western Blotting

• Whole cell lysates (WCLs) of eukaryotic cells
• SDS-PAGE
• Coomassie Blue staining of gels
• Western Blot: wet blot
• Western Blot: semi-dry blot
• Western Blot: probing of the membrane
• Western Blot: stripping of the membrane
• Dot Blot

Microscopy, Flow Cytometry and Histology

• Direct labelling of antibodies with fluorescent dyes
• Preparation of samples for confocal microscopy
• Immunofluorescence staining
• Immunofluorescence staining of focl adhesion proteins
• Live cell microscopy
• OPTIC (Opa protein triggered integrin clustering)
• Scanning electron microscopy (SEM)
• Staining of cells for flow cytometry
• Bacterial invasion assay by flow cytometry
• Bacterial invasion assay by flow cytometry
• Tissue/embryo preparation fo cryosectioning
• Fluorescent Staining of Cryosections

Molecular Biology

• Flow chart of a PCR-based cloning project
• Documentation and data storage for a novel recombinant plasmid DNA (cloning)
• Plasmid-Miniprep (Birnboim – Dooley protocol)
• Restriction digest of plasmid DNA
• Colony PCR
• Agarose gel electrophoresis
• Design principles for PCR primers used for amplification and cloning of cDNA
• PCR approach in 50 μL with hot-start and/or touch-down (preparative PCR)
• PCR approach in 20 μL with hot-start (qualitative PCR)
• Setting up quantitative real-time PCR qRT-PCR
• PCR purification
• Isolation of DNA from agarose gel
• Designing PCR-Primers for LIC Cloning (Ligation independent cloning)
• LIC Cloning (Ligation independent cloning)
• Cloning of shRNA into lentiviral vector pLKO.1
• Design of guideRNA-Oligos (gRNA) for CRISPR/Cas gen. editing
• Cloning of guideRNA-Oligo into pBluescript_U6-MCS
• Cloning of guideRNA-Oligo into pLentiCRISPRv2
• Site directed mutagenesis
• Cre-Lox recombination of plasmids
• InFusion-cloning (Fa. Clontech)

Microbiology and Protein Expression in E. coli

• Transformation of plasmids in E. coli
• Transformation of Neisseria gonorrhoeae
• Monitoring bacterial growth
• Gentamicin Protection Assay
• FBA-staining of bacteria
• Opsonizing bacteria with Fc fusion proteins
• Bacterial Pulldown with soluble receptors
• Expression of GST-fusion proteins
• Purification of GST-fusion proteins

Protein Expression in Pichia pastoris

• Generation of competent Pichia pastoris
• Transformation of Pichia pastoris
• Testexpression in Pichia pastoris