Aug 10, 2020
Preparation of staphylococcal protein-A conjugated to horseradish peroxidate by the periodate method.
- 1University of the West Indies St. Augustine
Protocol Citation: Angel A Justiz-Vaillant 2020. Preparation of staphylococcal protein-A conjugated to horseradish peroxidate by the periodate method.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjg8kjzw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 09, 2020
Last Modified: August 10, 2020
Protocol Integer ID: 40192
Abstract
This reagent can be used in ELISA, Western blotting and Dot blot to detect antigens and antibodies. It is important in the immunodiagnosis of infectious diseases and other problems. I find this useful in the detection of anti-HIV antibodies by ELISA.
Materials
MATERIALS
Ammonium SulfateP212121
Sodium periodateBio Basic Inc.Catalog #SB0875.SIZE.100g
sodium borohydrideSigma AldrichCatalog #452882
Horseradish Peroxidase (HRP) type IVSigma AldrichCatalog #P8375-25KU
Staphylococcal Protein-ASigma Aldrich
Pipettes
20ml to 1000 ml glass
Scale
Incubator
Refrigerator
Freezer
Centrifuges
Safety warnings
Pay attention to all details as the times of reactions among the proteins involved in this preparation. It will prevent over-oxidation. The average time of preparation is 18 hours.
Before start
All reagents but specially the enzyme and more importantly the sodium periodate solution has to be prepared freshly before mixing it with the enzyme.
Horseradish peroxidase (500 µg in 50 µl NaCO3 , pH 9.6) is mixed with freshly made sodium periodate solution (1.71 mg/ml) followed by incubation in the dark for 2 h.
Mix 500 µg of staphylococcal protein-A (SpA) with an equal amount (500 micrograms) of a mix of horseradish peroxidase-sodium periodate.
The mixture is incubated for 3 hours at 4°C with gentle agitation.
Forty µl of freshly prepared NaBH4 solution (5 mg NaBH4 /ml 0.1 mM NaOH) is then added to the preparation.
The preparation is incubated for 90 min at 4°C in the dark with gentle agitation.
Cold 50% saturated ammonium sulphate solution (pH 7.4) is added drop by drop in the ratio 1:1 (v/v).
The mixture is then centrifuged for 25 min at 4°C and recover the pellet at the bottom of the tube.
The pellets is re-suspended in 200 µl of PBS pH=7.4 and dialysed against 1L of PBS for 24 h with 3 buffer changes.
An equal volume of glycerol is added to the dialysate followed by 100 µl of bovine serum albumin, BSA (20 mg/ ml).
The conjugate is then stored at -20°C until further used.