Chapter 11: Analysis of Gene Expression: Translation

Analysis or protein abundance: biochemical approach → protein purification by chromatography → obtain amino acid sequence and develop DNA probes to screen cDNA for a clone

Proteome: Protein complement of a cell

Protein Purification

  • Must purify protein of interest from a mixture, commonly by column chromatography based on size (gel filtration), charge (ion exchange), antibody affinity etc.

SDS Page

Separation of denatured proteins based on size, useful to evaluate purte/composition of protein mixture and estimate approximate MW of a polypeptide, can be used in DNA sequencing for fragments close in size

  • Proteins are denatured and given a consistent charge by:
  • Boiling the samples in presence of heat and
  • reducing agent (betamerc or DTT) which reduces SH bonds to give a uniform negative charge
  • ionic detergent (SDS) which denatures the proteins and binds per unit mass to the protein
  • Make a standard curve on semi-log paper and plot distance migrated vs. size of protein markers extrapolate for your protein of interest
  • Proteins are electrophoresed through acrylamide gels which resolves polypeptides of different sizes with a sieving effect which separates proteins by MW, high concentration resolves smaller molecular weight
  • Bands on gel are visualized with Coomassie blue (not fine enough to detect contamination)
  • Comparing Abundance: Must load same amount of total protein and assay total protein in each sample
  • Assaying Protein Concentration: Add Coomassie blue to aliquot of protein sample, dye turns blue when binds protein and can be measure at specific wave length in a spectrophotometer, estimate concentration using standard curve of known protein concentration vs absorbance

Easy, cheap, analyze multiple samples, adjust size ranges, cut bands out of gel to sequence but each band may be mixture of similar proteins, spectrum depends on [gel] & protein abundance, can’t see native protein composition

2D Gels: First separate on native charge and then denature and separate by size, can detect members of gene family or find post-translational modifications

  • First Dimension: Apply protein to thin tube of pH gradient, protein reaches pI (if known specify pH range)
  • After proteins at pI, stop the gel and incubate IEF gel in SDS to denature and lay this on SDS gel
  • Second Dimension: Separate based on size/mass and see dots not bands, consider the % of acrylamide depending on what we want to resolve
  • Controls: look for good resolution of many protein spots, and compare to similar analysis

Can be used to compare proteomes, data shared via web, spots can be cut & sequenced, allows related forms to be distinguished but difficult for membrane proteins, proteome detected depends on extraction method & gel condition, highly abundant proteins make large spots that distort/hide other spots

Protein Detection Methods: Western/Immunoblot, Protein Fusions, Immunofluorescence, Immunolocalization

Western / Immunoblotting

  • Use of antibodies to detect proteins – incubation with antibody (binding reaction not hybridization)
  • Used to detect/identify specific protein in mixture and compare relative abundance in different mixtures
  • Antibodies can be used to detect mixtures separated by SDS PAGE or 2D GEL
  • After SDS PAGE transfer to membrane and incubate with antibodies, load same amount of protein and check if protein present (normalize to actin which will be present in equal amounts)
  • Primary antibody detects variable region (tag), secondary detects constant region of primary
  • Need sensitive detection (enzyme linked color or light / radioactivity)
  • To compare protein abundance, load equal amounts

Controls

  • Use protein extract on gel that has epitope recognized by antibody (dilution series to test antibody)
  • Include pre-stained proteins standard to estimate size of bound protein
  • Probe for actin to show equal protein loading, just ensure they are different sizes
  • Coomassie blue to stain gel for total protein


Very specific and sensitive, can be done on 2D gel as well but cannot be recovered, very low abundance may be difficult to detect (other ways to enhance primary antibody in this case)

To Quantify: Scan the membrane and count pixels to quantify then compare to acting

Analysis of Protein Subcellular Localization

  • Immunodetection is used to look at protein localization at different resolutions, cells, or subcellular levels
  • Location can reveal function, to determine location tissue must remain intact & localization determined by
  • Translation Fusion Protein - Transforming organism with transgene expressing protein of interest fused in frame (remove STOP) with a fluorescent protein
  • Incorporate epitope tag for commercial antibody into polypeptide, or incorporate GFP ORF
  • cDNA for protein is cloned in frame with tag under the control of promoter, transform host, watch movement and interactions of protein
  • Controls: Prove tag is functionally normal (functional complementation)
  • Detect specifically, versatile, localize more than 1 but need to ensure no impact of tag and transformable organism (transient transfections in some cases)
  • In Situ Immunofluorescence - Use antibody specific for protein of interest on tissue and react with detectable secondary antibody conjugated to FITC
  • Immunolocalization: Use antibody on very thin killed tissue and detect secondary antibody conjugated to gold ball with electron microscopy
  • Tissue is fixed with chemicals / cold to prevent cellular breakdown and embed in plastic, microtome prepares thin section of tissue and then incubated with antibody, washed and then secondary antibody conjugated to a gold ball, wash again, gold is electron dense can be viewed by electron microscopy
  • Controls: check antibody specificity using western blot,
  • -ve: use pre-immune serum from animal, omit primary antibody, incubate antibody and protein before adding to tissue, test with mutant without protein
  • +ve: test binding on mutant over expressing target protein, repeat many times
  • Very high resolution, specific way to detect, informative, estimate abundance, colocalize multiple (antibody from different animals), but time consuming, expensive, a lot of antibody, only binds to surface so difficult to detect low abundance, may not recognize native state (tag folded in)

Protein Profiling (Proteomics): Vary in composition, charge, size, so difficult. Each project unique in extraction, separation and quantification. Most common separation is 2D, HPLC Mass Spec, Gas Chromatography mass spec. Useful for post-translational modifications

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