Customer Comments

ImmunoPCR conjugates: Antibody/Oligo Conjugation

The major hurdle to iPCR chemistry was reliable, efficient and reproducible methods to prepare oligonucleotide/antibody conjugates. In one case a researcher was developing a second generation method that was more sensitive than the methods originally described 1 however using existing conjugation methods to prepare oligo/antibody conjugates purification was difficult and the yields were low.

Solulink was contracted to prepare multiple antibody/protein conjugates. Solulink delivered antibody/oligo conjugates in `~50% yield, >95% free from unconjugated oligo and antibody. These conjugates performed as required in the assays and many conjugates were subsequently prepared. In many cases only 100 ug of antibody was supplied and the team at Solulink was able to deliver functional conjugates- not a trivial task. A number of papers have been published wherein oligo/antibody conjugates were prepared using HydraLink bioconjugation technology by the researchers themselves 2,3 or under contract by Solulink 4,5.

• Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Gústafsdóttir SM, Ostman A, Landegren U.. Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol. 2002 May 473-7
• Simon Fredriksson, William Dixon, Hanlee Ji, Albert C Koong, Michael Mindrinos and Ronald W Davis, Multiplexed protein detection by proximity ligation for cancer biomarker validation , Nature Methods 4, 327, 2007
• Malin Jarvius*, Janna Paulsson, Irene Weibrecht, Karl-Johan Leuchowius, Ann-Catrin, Andersson, Carolina Wählby, Mats Gullberg, Johan Botling, Tobias Sjöblom, Boyka, Markova, Arne Östman, Ulf Landegren & Ola Söderberg, In situ detection of phosphorylated PDGF receptor β using a generalized proximity ligation method, Mol. And Cell. Proteomics 2007.
• Edith Schallmeiner, Elli Oksanen, Olle Ericsson, Lena Spångberg, Susann Eriksson, Ulf-Håkan Stenman, Kim Pettersson and Ulf Landegren, Sensitive protein detection via triple-binder proximity ligation assays, Nature Methods 2007 4, 1 35 – 137.
• Gali Steinberg-Tatman, Michael Huynh, David Barker, and Chanfeng Zhao, Bioconjugate Chem., 2006 841 – 848, Synthetic Modification of Silica Beads That Allows for Sequential Attachment of Two Different Oligonucleotides.
• I.A. Kozlov, P.C. Melnyk, K.E. Stromsborg, M.S.Chee, D.L. Barker and C. Zhao, Efficient Strategies for the Conjugation of Oligonucleoitdes to Antibodies Enabling Highly Sensitive Protein Detection, Biopolymers, 2004; 73(5):621-30.

Product used:

S-HyNic Conjugation Kit

Peptide / Magnetic bead conjugates

"Can I link my 40mer peptide to beads using your chemistry?" was the question from a Post Doctoral Fellow. "No," I said, "because if you use the lysines on a peptide to conjugate it to beads, it will compromise the epitope. However, by having the peptide re-synthesized with the HyNic linker added on the N-terminus during solid phase synthesis and simply adding the HyNic modified peptide to Solulink's 4FB-NanoLink beads you will have peoptide immobilized on magnetic beads."

6-Boc-HNA was ordered and the HyNic peptide was synthesized at the institute's peptide core facility. As advertised, the HyNic-peptide conjugated efficiently to the 4FB-NanoLink beads and the post doc was able to move her research foward within two weeks of ordering 6-Boc-HNA.

Products used:

Boc-HNA

NanoLink 4FB-Magnetic Microspheres

SPR and Solulink

“I have been trying to link my peptide to a SPR surface using EDC/NHS chemistry but the immobilization has been low yielding and not reproducible resulting in poor kinetic data. Can I link peptides to SPR surfaces using your chemistry without affecting the epitope?” was the question from a post-doctoral fellow that was studying the kinetics of binding of protein
to a peptide.

“Yes you can accomplish this in three straightforward steps. First have your peptide synthesized with our HyNic linker on the desired terminus, second prepare a 4FB surface by modifying an amino surface with sulfo-S-4FB and finally expose the 4FB surface to the HyNic peptide at
pH 5.0.”

The HyNic peptide was ordered from Solulink as well the sulfo-S-4FB and word came back from the post-doc that this worked as advertised and for the first time was able to get reproducible kinetic data.

Products used:

sulfo-S-4FB

S-HyNic

Contract Conjugation Services

Solulink’s Contract Conjugation Services has been contracted by scientists worldwide in both industry and academia for over seven years and has been asked to prepare a wide range of conjugates. In many cases clients had found that Solulink was the only company offering services that to prepare their conjugates. Some examples of conjugates that Solulink has successfully prepared include:

“In our hands, Solulink linkers have proven flexible and soundly designed products well tailored to customized production of bioconjugates.  The conjugation chemistry has wide application, is simple to perform, stable in solution and can be conveniently prepared in stock for later usage.    Solulink products are a valuable component of our laboratory toolbox.”

John Mountzouris, Ph.D.
Abgent

“I was extremely satisfied by how easily everything went together and based on these results will be hard-pressed to ever again use a maleimide.   This feedback is from someone who has tried all the other commercial coupling chemistries and none work so nicely as yours.”

Jim Coull
Ensemble Discovery

“We recently were able to link our TLR agonist to a phospolipid via your Solulink method. These hybrid molecules can be incorporated into nanoparticle formulations with the great advantage that the incorporation of the conjugate into the nanoparticle can be quantified via UV absorbance mearurements which do not interfere with the other components of the complex particle. I highly recommend your linker method for this application.”

Wolfgang Wrasidlo PhD
University of California in San Diego

“We have successfully used the versatile linker, SANH (Succinimidyl 6-hydrazinonicotinate acetone hydrazone), to modify proteins of interest, namely mouse serum albumin and ovalbumin, and subsequently conjugate the intermediate to a small molecule containing an aromatic aldehyde to form a stable hydrazone product.  The reactions were found to be reproducible and convenient to perform.  The final products were easily characterized by UV spectrophotometry to determine the extent of conjugation.  SANH is the most versatile and convenient linker reagent we have found for our protein-small molecule modifications.”

Howard B. Cottam, Ph.D.
Moores Cancer Center

SoluLinK - The Conjugation Company
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San Diego, California 92121