The Antibody-Oligonucleotide All-in-One Conjugation Kit uses patented SoluLINK® bioconjugation technology to link an antibody to an oligonucleotide. A succinimidyl-4-formylbenzamide (S-4FB)-modified oligonucleotide and succinimidyl-6-hydrazino-nicotinamide (S-HyNic)-modified antibody form an antibody-oligonucleotide conjugate. This kit is specifically designed to conjugate 100 µg of antibody to any amino-modified oligonucleotide in the 20-80 nucleotide range.
Any mammalian monoclonal or polyclonal IgG isotype antibody can be conjugated to the oligonucleotide and purified with approximately 4 hours of total hands-on time.
SoluLINK bioconjugation technology forms a stable and UV-traceable bis-aryl hydrazone bond with measurable absorbance at 354 nm, which has a molar extinction coefficient of 29,000 L mol-1 cm-1.
The conjugation reaction is spectrophotometrically quantifiable due to the formation of a chromophoric conjugate bond.
The Antibody-Oligonucleotide All-in-One Conjugation kit uses the SoluLINK patented conjugation technology to link an antibody to an oligonucleotide, as illustrated in Figure 1. The first stage of the process begins with the labeling of a 3’ or 5’- amino-modified oligonucleotide withS-4FB. This amine-reactive NHS ester incorporates an aromatic aldehyde functional group, formylbenzamide (4FB), at the desired terminus of the oligonucleotide.
Figure 1. Reaction of a HyNic-modified IgG with a 4FB-modified oligo leads to the rapid formation of a stable antibody-oligonucleotide conjugate.
In stage two of the process, a polyclonal or monoclonal antibody (100 μg) is modified using the complementary linker S-HyNic. This NHS-ester reacts with lysine residues, incorporating HyNic functional groups (hydrazinonicotinamide) onto the antibody. In the third and final stage, the two modified biomolecules are mixed together in the presence of a reaction catalyst (aniline) to form the conjugate, after which purification is carried out using a magnetic affinity solid phase.
2) Conjugate Purification
Antibody-oligonucleotide conjugates produced with the All-in-One kit are ready to be used in the most demanding and sensitive of downstream applications. The kit delivers high-purity conjugate virtually free of residual antibody or oligonucleotide (>98%). Reaction conditions are optimized to convert nearly 100% of the antibody into conjugate, leaving only free, excess 4FB-oligo to be removed. Complete conversion of antibody to conjugate simplifies conjugate purification as illustrated in Figure 2. Antibody-oligonucleotide conjugate is purified to near homogeneity by selectively binding the conjugate to a magnetic affinity matrix, allowing excess 4FB-oligonucleotide to be washed away. Affinity-bound conjugate is then gently eluted from the matrix and buffer exchanged into long-term storage buffer. Antibody-oligonucleotide conjugates produced with this kit are stable for up to 1 year when kept at 4oC in storage buffer.
* 4FB MSR is an acronym for 4-formylbenzamide molar substitution ratio.
Figure 3. Stage 1 of the process illustrates the modification of an amino-oligonucleotide with the S-4FB linker.
Figure 4. Stage 2 of the process illustrates the modification of IgG using the S-HyNic linker.
Figure 5. Stage 3 of the process illustrates both the formation and purification of the conjugate.
SoluLINK® Bioconjugation For Research Use Only. Not for use in diagnostic procedures. For additional licensing restrictions, please see the license agreement at vectorlabs.com/solulink-research-license.
Products are for research use only, not for use in diagnostic or therapeutic procedures or for use in humans. Products are not for resale without express written permission of Seller. No license under any patent or other intellectual property right of Seller or its licensors is granted or implied by the purchase unless otherwise provided in writing.