Lab Protocols and Reagents

Protocols

The following protocols are commonly utilized in our lab.

In Vivo Experimentation

  • Targeted genetic manipulation of mice
  • Breeding and genotyping mice with various mutations
  • Genetic, dietary and chemical models of obesity, diabetes and atherosclerosis
  • Metabolic characterization of mice, indirect calorimetry, DEXA
  • Clamp Studies
  • Athesclerosis Studies
  • Isolation of primary cells and establishment of cell lines

Cell Culture

  • Thawing, propagating and freezing cell lines
  • Serum preparations for special applications
  • Differentiation of pre-adipocyte cell lines
  • Cholesterol uptake and efflux
  • Insulin signaling in differentiated adipocytes
  • Deoxy-glucose uptake in preadipocytes and in differentiated adipocytes
  • Stimulated lipoysis in differentiated adipocytes
  • Co-culture protocols for differentiated adipocytes and other cells
  • Metabolic labeling (35S – 32P)
  • Immunocytochemistry

Protein Biochemistry

  • Protein isolation from whole tissues and cultured cells
  • Subcellular fractionation of tissue samples and cells
  • Recombinant protein production, quantitation (Bacterial, Mammalian)
  • Fusion proteins
  • Affinity chromatography
  • SDS-PAGE
  • Immunoblotting, immunoprecipitaion
  • In vitro kinase assays

Molecular Biology

  • Total RNA isolation from whole tissues and cultured cells
  • DNA isolation from whole tissues and cultured cells
  • Northern-blotting
  • Southern blotting, RT-PCR
  • Mutagenesis
  • Subcloning (vectors, enzymes)
  • Ligation (blunt-end, sticky-end)
  • Sequence analysis
  • Agarose gel electrophoresis (regular, high resolution)
  • Transient transfection
  • Retroviral transfection/infection
  • Adenoviral gene transfer
  • Lentiviral gene transfer
  • Luciferase reporter assays
  • Mobility shift assays, DNAseI footprinting
  • RNAi mediated gene regulation

Reagent Collection

The collection of biological reagents available in our lab include the following:

Vectors and cDNA clones

Expression/genomic libraries

  • cDNA libraries from white and brown adipocytes
  • Yeast-2 hybrid library from white adipose tissue

Cell lines

  • Murine pre-adipocytes(e.g. 3T3-L1,F442A,TNFR1-/-,TNFR2-/-, TNFR1-/-R2-/-, aP2-/-, mal1-/-)
  • Packaging cells (e.g. Bosc) and many more
  • Monocyte/macrophage (RAW, U937, THP-1, aP2-/-, mal1-/-, LXR-/-, PPAR-/-)
  • Primary macrophages (splenic/bone marrow/thioglycollate ellicited)
  • Primary and established MEF cells from various mutants
  • Antibodies

Murine mutant models

  • Various Cre Models
  • GATA2-LoxP
  • GATA3-LoxP
  • GATA2/3-LoxP
  • STAMP1-/-
  • STAMP2-/-
  • TNF alpha-/-, ob/ob & TNF alpha-/-
  • TNFR1-/-, TNFR2-/-, TNFR1-/-R2-/- and all in ob/ob background
  • JNK1-/-, JNK2-/-, JNK3-/-, XBP1-/+
  • aP2-/-, mal1-/-, aP2-mal1-/- and all in ob/ob and ApoE-/- backgrounds
  • ob/ob, db/db
  • yellow, tubby