Applications

Applications

Dot-it-Spot-it® Protein Assay

2015-09-16

General use of Dot-it-Spot-it Protein Assay

For all protein assays you will need to select a reference protein for use as calibration curve.
The reference protein has to be diluted in the same buffer as you have your samples in.

For protein concentrations below 0.1 mg/mL there will be a substantial loss of protein to surfaces like tube walls.
Detergents are commonly added to reduce the loss of protein.

1. Calibration curve

Calibrate with a protein or protein mixture that corresponds to your samples.
The concentration of pure proteins can be checked by their absorbance at 280 nm, using a 1 cm pathway in the photometer.
1 mg/mL BSA = 0.67 AU
1 mg/mL HSA= 0.53 AU
1 mg/mL IgG = 1.37 AU

2. Dilution buffer

  • Dilute your calibration protein in the same buffer as you have your samples in.
  • If you have to dilute the samples, a buffer containing 1% SDS, with phosphate or Tris buffered saline, will be suitable. Use the same final buffer composition for diluting your calibration protein.

SDS: Sodium dodecyl sulfate

2015-09-16  Using Dot-it-Spot-it for quantification during and after: 

Extraction of cells and tissue samples

Dot-it-Spot-it analysis in the nanogram range have been performed with these extraction buffers:

  • 1% OGP, 10 mM TRIS-HCl pH 7.5, 0.15 M NaCl, 1 mM EDTA.
  • 1% OGP, 0.1 M TEAB, 20% ACN, 5 mM DTT.
  • 0.1% RG, 0.1 M TEAB, 20% ACN, 5 mM DTT.

OGP: Octyl-beta-D-Glucopyranoside
RG: RapiGest SF surfactant, Waters
TEAB: Triethylammoniumbicarbonate
ACN: Acetonitrile
DTT: Dithiothreitol
EDTA: Ethylenediaminetetraacetic acid

2015-09-16  Using Dot-it-Spot-it for quantification after:

Electrophoresis

pI estimation

IEF (isoelectric focusing) was performed with the OFFGEL Fractionator, Agilent.

The 24 fractions obtained after the IEF separation were analysed with Dot-it-Spot-it, taking out only nanogram of protein. The electrophoresis buffer (5% glycerol, 1% tween 20, 0.3% OFFGEL buffer 3-10 ampholyte) was 1:2 diluted with 1% SDS, 20 mM TRIS-HCl pH 7.5, 0.15 M NaCl before Dot-it-Spot-it analysis. The pI (isoelectric point) of protein, protein isoforms or protein mixture, was estimated by combining the rapid quantitative estimation of protein distribution with the measurement of pH in the fractions.