Aug 11, 2020
Protocol of preparation of a protein-LAG conjugated to horseradish peroxidase by the periodate method.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Protocol of preparation of a protein-LAG conjugated to horseradish peroxidase by the periodate method.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjk7kkzn
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 11, 2020
Last Modified: August 11, 2020
Protocol Integer ID: 40319
Abstract
Peroxidase conjugated to proteins L, A and G (SpLAG-HRP) is a new development in the field of biochemistry. This versatile protein binds to more 500 species of animal IgGs. It makes it suitable for the study of zoonosis and the assessment of the antibody production to infectious microorganisms. This immunomarker is not available in a recombinant form as their counterpart SpLA-HRP, SpLG-HRP and SpAG-HRP. It can be used in ELISA, immunohistochemistry, Western blot analysis, Dot blot, and RIA [1,2].
References
1. Justiz-Vaillant AA, Akpaka PE, McFarlane-Anderson N, Smikle MF. Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species.West Indian Med J. 2013;62(1):12-20.
2. Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175.
Guidelines
All reagents but specially the enzyme and the sodium periodate solution have to be prepared freshly before mixing it with the enzyme.
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
Protein-L from P. Magnus
Streptococcal protein G by Sigma Aldrich
Pipettes
20ml to 1000 ml glass
Scale
Incubator
Refrigerator
Freezer
Centrifuges
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.
Mix 500 µg of streptococcal protein-G (SpG) with an equal amount (500 micrograms) of a mix of horseradish peroxidase-sodium periodate.
Mix 500 µg of Peptostreptococcal protein-L (SpL) with an equal amount (500 micrograms) of a mix of horseradish peroxidase-sodium periodate.
The three mixtures are incubated separately 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 each mixture and then, the preparations are centrifuged (13,000rpm., 10 minutes at RT). Add to each preparation cold saturated amonium sulphate solution and centrifuge them again (10000rpm, 25 minutes at 4°C).
Mix the 3 preparations and incubate in the dark with gentle agitation.
The mixture is then centrifuged for 25 min at 4°C and recover the pellet at the bottom of the tube.
The pellet (SpLAG-HRP) is re-suspended in 500 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 200 µl of bovine serum albumin, BSA (20 mg/ ml).
The SpLAG-HRP conjugate is then stored at -20°C until further used.