Protein Assay

Protein Assay

1. Uses

   • Defintion
     • Protein
       • Organic compound containing nitrogen
     • Micro
       • Small
     • Array
       • arrange in an order
   • 1. Antibody Arrays – Used to detect proteins or antibodies present in the sample
   • 2. Sandwich Arrays – Molecule detection by use of a ‘Sandwich of antibodies’
   • 3. Protein / Epitope Array – Proteins or protein fragments bound directly to the array surface

2. Sample Detection

   • Hybridisation used to bind the primary antibody with the sample (e.g. milk)
   • Secondary antibody (for primary antibody detection) that contains and fluorescence conjugated
   • Incubation of Secondary antibody-Flurophore and primary-sample
   • Microarray Fluorescent Scanner used for detection

3. Data Correction and Analysis

   • Separate data expressed for each wavelength
     • Fluorescence value (F) = Average intensity within spot area
     • Background Value (B) = Average intensity outside of spot area
     • Repeated three times to get average net intensity
   • With samples that have Autofluorescence a control must be tested first in which there are no antibodies added.
   • Visualising results
     • Bar charts
     • Cluster analysis graphs
   • Normalisation = Final normalization allows you to be able to compare the results by slight elimination of variations
   • When using Fluorescence spill over (using multiple dyes in one experiment) a spillover coefficient is needed

4. Technique Validation

   • Definition : Before publishing your results you must first test using other methods if your data is correct or not
   • If changes shown by the protein microarray can be confirmed using similar techniques then the hypothesises can be critically tested
   • If changes shown by the protein microarray can be confirmed using similar techniques then the hypothesises can be critically tested
   • Definition : before publishing data you must first test if your data correlates with other similar methods

5. Sample Collection

   • 1. Sample collected and split into:
     • Crude = containing DNA fragments, lipid and protein
     • Pure = containing protein
   • 2. Several Reaction to Purify the protein solution
   • 3. Blocking of slides to ensure that the proteins bind only to the Printed Spots and not the Nitrocellulose surface
   • 4. Printed on slides by robots and left to dry

Images and files in the map