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Wednesday, December 4, 2013

Protein Quantification / Protein Estimation using Polyacrylamide gel Electrophoresis (PAGE)


The Sodium Dodecyl Sulfate -Polyacrylamide Gel Electrophoresis (SDS-PAGE) is a technique for the characterization of proteins both quantitatively and qualitatively. It is a separation technique to separate out proteins from a mixture based on the molecular weight. SDS- PAGE can also be used quantify  particular proteins at microgram level from a mixture. The quantification analysis can be done by scanning the stained electrophoresed gel by densitometry. If the polypeptides are radiolabelled then it can be visualized and quantified using flourographic plate.

Principle
The percent absorption of incident light is directly proportional to the color intensity of the protein-dye complex on the gel and is directly related to the protein concentration.
Similarly, the intensity of darkening of the X-ray plate is directly proportional to the radioactivity in the protein in fluorographic plates.

Materials
  • A spectrophotometer with suitable scanning facility and chart recorder (or integerator facility, if available)
  • Protein Stain (Quantitative): 0.2% Proceion Navy MXRB dye in Methanol : Acetic Acid : Water (5:1:4). Dissolve the dye first in the methanol and then proceed. Prepare fresh everytime.
  • Destaining Solution: Methanol : Acetic Acid : Water (1:1:8).
  • Fluorograph Plate - For Fluorograph Scan
Procedure
  1. 1. After the electrophoresis (SDS-PAGE of protein), immerse the gel in Proceion Navy dye solution and shake until the proteins are completely stained (for a fixed period ~ 2hrs).
  2. Destain the gel until the background is colorless.
  3. Scan the gel at 580nm to measure the degree of dye bound by each band of protein. Depending upon the type of equipment available for scanning, the whole gel is used or each lane is cut out and scanned individually. The total absorption by the dye in each band is proportional to the area of the peak in the scan profile.
  4. Each peak in the scan profile is traced using a planimeter to determine the area under it. Otherwise, each peak in the chart may be cut out and weighed. When an integrator is interposed, the area under each peak is automatically calculated. 
  5. A curve is obtained by plotting A580 vs. amount of protein used as standard. Bovine serum albumin (Fraction V) at different known concentrations co-electrophoresed in different lanes in the same gel is also used to construct the standard curve. It should however be noted that the protein both in the standard and under examination to have equal dye-binding property.
  6. Scanning Fluorographic Plate
  7. Scan the individual lane strip or the whole fluorographic plate at 620nm as described above. The standard curve is obtaining using a radioactivity labeled standard protein whose concentration and radioactivity are known.
Notes

The following conditions need to be satisfied to quantity proteins on the gels:
  • The protein bands should be well resolved,
  • The dye should be bind to the protein of interest, and the binding should be uniform to all proteins and the sampling errors should be small.
  1. If the peaks are not well resolved, use of the narrower beam of light will improve the situation but at the cost of baseline.
  2. Coomassie brilliant blue R250 staining is not suitable for quantitative analysis of proteins although it is a highly sensitive stain. 
  3. Proceion Navy dye binds to the proteins stochiometrically and covalently. Destaining of this dye from the gel requires longer time.
  4. Sampling errors are inevitable but their effect can be reduced by repetition and averaging the results.
  5. Electrophoresis with a fixed sample volume, voltage and duration of run is necessary between runs to obtain satisfactory results.
  6. Gel scanner with computer facility are now commercially available.
References
1. Carlier, A R, Manickam, A and Peumans, W J (1980) Planta 149 227.
2. Smith, B J, Toogood, C and Johns, E W (1980) J Chromatogr 200 200.