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Avidity
BIS300
详细说明
BRTA (Biotinylation Reaction Titration Assay):
This is an ELISA-like reaction using BIS-300 kit components to examine the extent of biotinylation of an
AviTag’d protein-of-interest versus a known amount of biotinylated standard. The BRTA procedure
follows a typical ELISA protocol. The standard protein and unknown proteins are adsorbed to the wells
of a 96-well plate at known protein quantities. The biotin associated with the AviTag™ is detected by its
interaction with streptavidin-conjugated alkaline phosphatase. After adsorption, extraneous biotin is
removed by washing the wells, eliminating the need for dialysis steps. NOTE: Some test proteins may
not bind efficiently to the plastic of the 96-well plate.
Reagents needed:
? Fully biotinylated MBP-AviTag fusion protein standard (1mg/ml biotinylated MBP-AviTag™; BIS-
300 kit component; Avidity, LLC)
? Un-biotinylated MBP-AviTag fusion protein (1mg/ml unbiotinylated MBP-AviTag™; BIS-300 kit
component; Avidity, LLC)
? Streptavidin-Alkaline Phosphatase conjugate (Molecular Probes or other)
Solutions:
? PBS: 138mM NaCl, 2.6mM KCl in 10mM potassium phosphate (pH 7.4)
? PBST: PBS plus 0.05% Tween 20
? TBS: 10mM Tris, pH 7.5, 150mM NaCl
? Blocking Solution: PBS plus 40μg/mL BSA
? Dilution Buffer: PBS plus 0.15μg/mL BSA
? Streptavidin-Alkaline Phosphatase conjugate solution: 40μg/mL in PBS
? Development Solution for alkaline phosphatase: p-nitrophenyl phosphate in diethanolamine
buffer (Bio-Rad Laboratories)
? Stop Buffer: 2M KOH
Equipment /materials:
? 96-well polystyrene, high binding, flat bottom micro-titer plates (Corning Costar Corp.)
? Biokinetics Reader EL312e plate reader (Bio-Tek Instruments) or other.
Protocol:
1. Use one 96-well polystyrene micro-titer plate for all of the following:
? Standard curve: coat ten (10) wells with 50μL each of 1 to 10ng (in 1ng increments) fully
biotinylated MBP-AviTag™ fusion protein diluted in Dilution Buffer. Coat seven (7) additional
wells with 50μL each of 15 to 45ng (in 5ng increments) fully biotinylated MBP-AviTag™ fusion
protein diluted in Dilution Buffer.
? Biotinylated sample: coat new wells on the same plate as done above with 1 to 45ng of the
biotinylated protein-of-interest in Dilution Buffer. The degree of purity of the test protein
samples must be determined to accurately assess the degree of biotinylation. For example, if
the protein-of-interest is only 80% of the total protein in the sample, this must be accounted for
when making the dilutions.
? Negative control/blanks: add 45ng of the un-biotinylated MBP-AviTag fusion protein in Dilution
Buffer to a single well as a negative control. Also add wells containing Dilution Buffer alone to
act as blanks for the plate reader.
2. Allow the proteins to adsorb to the plate for at least 1 hour at room temperature with gentle
rotational shaking.
3. Shake the liquid out of the wells over a sink and wash the plate wells four times with PBST.
4. Incubate plate with 300μL of Blocking Solution in all the assay wells for 1 hour at room
temperature with gentle rotational shaking to block non-specific binding.
5. Shake the liquid out of the wells over a sink and wash the plate wells four times with PBST.
6. Pipette 50μL of the 40μg/mL streptavidin-alkaline phosphatase conjugate solution into each
assay well and incubate with gentle rotational shaking for at least 1 hour at room temperature.
7. Shake the liquid out of the wells over a sink and wash the plate wells four times with PBST.
