Dot-it-Spot-it® Protein Assay
General use of Dot-it-Spot-it Total Protein Assay
The Dot-it-Spot-it Total Protein Assay is an ultrasensitive test, using very low amounts of protein. When you are working with 0.1 ng protein you need to be aware of some pitfalls.
1. Make sure that you have clean tubes, clean pipette tips, clean gloves!
- Plastic consumables, like tubes and tips, may be manufactured using slip agents and plasticizers that can to leach into the sample. We have found that some brands of polypropylene micro tubes affect the results. When we are working with a buffer containing 0.03% Tween 20, which reduce adsorption, we are using Forensic DNA Grade Eppendorf tubes, and Eppendorf epT.I.P.S, with good results. The tubes and tips are handled with clean gloves.
2. Do you generate additional proteins?
- There will be a nice fingerprint if you place your finger on the Detection sheet.
- Saliva contains about 1 mg protein per mL, so be careful with talking and laughing.
- If you works with protein stock solutions of 1 mg/mL, remember that the concentration is several thousands times higher than the concentration you measure, and your gloves will be contaminated.
- If you see dust, as very small black marks, on the scanned detection sheet – use a brush to remove dust particles before immersing the Detection sheet into the Detection solution. We use Kinetronics StaticWisk, an anti-static brush.
- For protein concentrations below 0.1 mg/mL there will be a substantial loss of protein to tube walls. Detergents, like Tween 20, are commonly added to reduce loss of protein.
- Proteolytical enzymes can in some seconds degrade the protein. Protease inhibitors, like EDTA, might be necessary to add.
- 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
Dilute your calibration protein in the same buffer as you have your samples in.
3. Do you have dust in the laboratory?
4. Avoid protein adsorption and protease activity.
5. The need for a calibration curve.
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.
RG: RapiGest SF surfactant, Waters
EDTA: Ethylenediaminetetraacetic acid
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.