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The coupling modification of gABA
gABA（Gamma-Aminobutyric Acid）is the main inhibitory neurotransmitter of
central nervous system in mammal, which mainly reduces the excitability of neurons.
Besides the ability of directly adjusting the muscle tensity, gABA also has other
functions, for instance, people will feel anxious and tired when he/she lacks gABA in
the body. Consequently, gABA has great influences on normal physiological function
of human being.
Fig 1 The molecular structure of gABA
gABA is a natural non-protein amino acid, which shares same molecular weight
in different species. Aiming at this, Cloud-Clone Corp. developed a series of
detection tools on gABA which includes coupling molecules, antibodies and Elisa
kits. The development and market access of gABA products bring researchers great
access on gABA research tools.
1.
The coupling modification of gABA
gABA is a small molecule that has no immunogenicity, which needs to couple
with other proteins to acquire immunogenicity. Cloud-Clone Corp. adopts
glutaraldehyde to couple gABA with BSA, OVA and HAS to obtain BSA Conjugated
gABA (CPA900Ge11), OVA Conjugated gABA (CPA900Ge21) and HSA Conjugated
gABA (CPA900Ge41). The process is as followed. Dissolving gABA and any of
above vector proteins in PBS, and then adding glutaraldehyde and reacting for 1 h,
followed by separating with G-100 column, antigen with immunogenicity was
obtained.
Fig 2 The principle of gABA coupling
2.
Preparation of anti-gABA antibody and its coupling modification
The OVA-gABA was used as the antigen to immunize rabbit to prepare
antiserum, after purification, specific anti-gABA polyclonal antibodies were obtained
(PAA900Ge01).
And then, the antibody was labeled by biotin to prepare biotin-linked antibody
to gABA (PAA900Ge71).
Biotin labeling method:
Fig 3 The molecular structure of biotin
There are two cyclic structures in biotin (Fig. 3), the valeric acid side chain end
is the binding site of antibody or other biomacromolecules. The biotin is activated by
chemical modification, and then the activated biotin could be coupled with
biomacromolecules
by
one
kind
of
protein
cross-linking
agent,
the
biomacromolecules include protein, nucleic acid, polysaccharide, lipid, etc.
Dissolving gABA with carbonate buffer solution, adding the same mole of
activated biotin-ester, mixing and depositing overnight at 37℃. After getting rid of
the uncombined gABA by dialysis tube with 0.01M PBS, the conjugated products
were collected, concentrated and freeze-dried for later use.
The FITC labeling method:
Fig 4 The molecular structure of FITC
In alkaline conditions, The sulfur producyion chemical bound of FITC will react
with gamma amino groups of lysine in protein to get FITC labeled fluorescent
compounds. The basic principle is as followed:
FITC-N=C=S +N-H2-protein
FITC-NS-C-N-H2-protein
Cloud-Clone Corp. adopts the Marsshall method to label FITC on gABA.
3. The gABA Elisa kits
Based on the development of coupled molecules and specific antibodies,
Cloud-Clone Corp. develops gABA Elisa kit (CEA900Ge). As gABA is a small
molecule and exists as the same form in different species, so the antibodies and kits
could be used in the detection of gABA from samples collected from different
species.
For more information, please visit http://www.cloud-clone.us/.