Aug 11, 2020
Preparation of a protein-LG 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. Preparation of a protein-LG conjugated to horseradish peroxidase by the periodate method.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjk3kkyn
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: 40315
Abstract
SpLG comprises of 4 Ig-binding domains of SpL and 2 IgG Fc-binding SpG domains [1]. This hybrid molecule was found to bind many intact human Ig molecules and Ig fragments [1]. It proved a powerful tool for the binding, detection and purification of antibodies [2].It was reported that chimeric SpLG was a potent mitogen for mouse splenic B cells, and induced cell differentiation and the production of immunoglobulins. Inhibition experiments demonstrated that the Ig-binding capacity of both SpG and SpL in the chimeric molecule are independent of each other. It was also shown that SpLG selectively absorbed Igs present in the sera of humans, rabbits, mice and rats [1-3]. The preparation of SpLG-HRP by the periodate method is a novel application [4].
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. Kihlberg BM, Sjöbring U, Kastern W, Björck L. Protein LG: a hybrid molecule with unique immunoglobulin binding properties. J Biol Chem 1992, 267(35): 25583-8.
3. Justiz-Vaillant AA, McFarlane-Anderson N, and Smikle M. “Bacterial Immunoglobulin (Ig)-Receptors: Past and Present Perspectives.” American Journal of Microbiological Research, vol. 5, no. 2 (2017): 44-50. doi: 10.12691/ajmr-5-2-4.
4. 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.
Materials
MATERIALS
Ammonium SulfateP212121
Sodium periodateBio Basic Inc.Catalog #SB0875.SIZE.100g
sodium borohydrideSigma AldrichCatalog #452882
Horseradish Peroxidase (HRP) type IVSigma AldrichCatalog #P8375-25KU
Protein-L from P. Magnus
Streptococcal protein G by Sigma 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 (SpL) with an equal amount (500 micrograms) of a mix of horseradish peroxidase-sodium periodate. On the other hand mix 500 µg of recombinant protein-L with an equal amount (500 micrograms) of the mix of horseradish peroxidase-sodium periodate.
The two 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 separately to the preparations, which are centrifuge (13,000rpm., 10 minutes at RT). Add to each preparation cold saturated amonium sulphate solution and centrifuge again (10000rpm, 25 minutes at 4°C).
Now mix the SpL-HRP preparation with SpG-HRP and incubate the mixture for 90 min at 4°C 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 (SpLG-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 conjugate is then stored at -20°C until further used.