We recently bought your technetium linker kit. The protocols are fairly well explained up until the process of linking technetium (in the form 99m-technetium pertechnetate) with your HyNic linker. We simply want to couple 99mTc pertechnetate to your HyNic linker (already bonded to a backbone substrate). Can you direct us to a simple protocol of how to incorporate the 99mTc pertechnetate into your HyNic protocol?

http://www.nucmedbio.com/article/S0969-8051%2803%2900056-8/abstract

 

Regarding the antibody-oligo conjugation…we are interested in conjugating oligos to smaller binding proteins that have a unique cysteine tag. Is there a protein-oligo kit available for conjugations to cysteines? If not, do you think it would work to use the MHPH (Maleimide HyNic) crosslinker and the S-4FB crosslinker for the amine-oligo to cys-protein conjugation? When reading the protocol for the MHPH, it does not say that the protein needs to be reduced, is this correct?

We do not have a protein-oligo kit made specifically for protein[cysteine]-oligo conjugations, but your planned strategy is exactly right. You would want to use the S-4FB crosslinker to modify your oligonucleotide, which has an amino group at the 3’ or 5’ end, and crosslink that product to an antibody (or protein) modified with HyNic. The way to conjugate your protein (at a Cys residue instead of a Lys residue) to HyNic is with our MHPH reagent, then react the two modified biomolecules together to get your conjugate. It is recommended to add TCEP (tricarboxyethylphosphine) to the MHPH modification reaction, which reduces any disulfide bonds, but does not react with the maleimido group. Recommended conditions are adding 3-5 mol equiv of TCEP/mol protein during the modification step. We also recommend conjugating the HyNic modified protein immediately.

I’m interested in using your Antibody-Oligo conjugation kit. One question that has come up already for me was what is meant by “amino-modified” for the oligo? Would a standard DNA synthesis company know exactly is meant by that or is it something special?

Starting Amino-Oligonucleotide requirements: The Antibody-Oligonucleotide All-in-One™ kit is designed to conjugate any high purity 5’ or 3’ amino-modified oligonucleotide (20-60 nucleotides in length) to any monoclonal or polyclonal IgG-class antibody. The protocol requires a minimum quantity of 10 OD260 and a maximum of 40 OD260 units of HPLC purified amino-oligonucleotide. Solulink recommends that longer oligo sequences (e.g., > 49-mer) be synthesized with a 5’-amino group and shorter oligos (< 49-mer) with a 3’-amino group if the specific application permits. Oligonucleotides <49 bases can be either reverse phase (RP) or ion exchange purified (IEX) while longer oligos (> 49-mer) can be IEX or double HPLC purified depending on the specific services offered by the vendor. Some vendors offer these purification options on a custom basis while others offer them as a standard service, albeit at additional cost. Be advised that unpurified 3’-amino oligos contain a significant quantity of truncated failure sequences that lead to undesirable conjugation products while unpurified 5’-amino oligos contain up to 50% of A260 units in the form of shorter unmodified failure sequences that never form conjugate and thereby alter the stoichiometry of the conjugation reaction. For best results always use the highest quality, HPLC purified amino-oligonucleotide available.
Note: Please be advised that some oligo vendors will not HPLC purify amino-modified oligos or in some cases longer oligonucleotide sequences (modified or unmodified) except as a custom service. However some oligo suppliers do offer these services as a standard option. TriLink recommends that customers always use HPLC purified amino-oligonucleotides in this protocol. We recommend requesting a mass spectrum to confirm the final quality when available. The mass spectrum confirms percent full-length purity as well as molecular weight (unambiguous confirmation of amino group). As a general rule, we do not recommend using crude oligonucleotide preparations to make a conjugate. Use barrier pipette tips and good laboratory practices at all times to avoid potential contamination and/or cross-talk between different oligonucleotide sequences.

How does the TriLink Antibody-Oligonucleotide kit differ from what Olink offers?

The following list describes how TriLink’s All-in-One Antibody/Oligonucleotide Conjugation Kit differs from what Olink offers:
Olink’s antibody/oligo product is a component of their in situ IHC/PLA product
It includes the specific oligos for that product
It conjugates only 10 µg of antibody
There is no indication of the purity, i.e., % conversion to conjugate
There is no purification step
It is not available as a standalone product

I want to use a cleavable 4FB linker with your antibody-oligo kit. Which is the cleavable linker?

If you use the Ab-oligo kit, this comes with the S-4FB crosslinker. Instead of using the crosslinker in the kit, you may use an alternative 4FB crosslinker to attach to your oligo, the S-SS-4FB crosslinker (Cat. No. S-1037). The Excel spreadsheet that comes with the kit (the calculator) can tell you how much of the reagent to use once resuspended. You would then be able to cleave the Ab-oligo linkage, which is a disulfide bond.

For your antibody-oligo kit, can conjugates be prepared with larger amounts of antibody?

You can prepare antibody-oligo conjugates in larger quantities. I would recommend buying multiple Ab-oligo kits (we have volume discounts), because they are much easier to use than any alternative. Alternatively, you can use the more general protein-oligo kit, which can conjugate mg amounts amount of antibody. But, this kit does not contain the unique purification step that comes in the Ab-oligo kit, and requires HPLC purification of the final antibody-oligo conjugate.

In the antibody-oligo kit, I wonder if, after the purification process (part B and C in the user manual), all of the unconjugated DNA oligo and antibody should be removed from the system? if so, can we use the total antibody concentration as the value for the coupling product?

The protocol is set up so there will be < 5% free oligo or free antibody. We recommend that you analyze your conjugates, at least initially, by gel electrophoresis. We recommend a 5–20% gradient gel developed with Coomassie (for protein) or silver stain (for oligo.) That will tell you how well the conjugations worked.

After the coupling reaction (antibody-oligo), we used a Nanodrop ND-1000 to test the absorbance of the coupling products. We first tested the absorbance at 354 nm (user manual); we wanted to confirm if the conjugation was successful. We did not observe strong absorbance there, so we tested the absorbance of the product.

The 354 absorbance is small compared to the oligo A260 but what you have is reasonable. You can quantify the number of oligos attached using the extinction coefficient of the oligo (from the product data sheet, molar extinction coefficient at A354 is 29,000). Note the protein does not contribute much to the A260 so it can be ignored unless you want to be very accurate. Determine the protein concentration with a BCA assay. With those numbers, you can accurately determine the number of oligos/antibody.

Can I use a longer oligo (longer than 60 bases) in your Antibody-Oligo kit?

In our experience, oligos up to 60 bases work best. If you use a longer oligo, we cannot guarantee success, but it should still work for you with some slight modification to the protocol. We would suggest that you allow the conjugation reaction to go overnight at 4ºC. This should improve the efficiency, which you might expect to be lower as the length of the oligo increases.

With plans to conjugate a protein to antibody, previous attempts, via amine linkage on one end (the antibody) and maleimide coupling on the other end (via a cysteine residue on my protein), the results were not optimal. More reproducible results and more control of the process is desired, specifically with less proteins conjugated to each antibody, ideally a 1:1 ratio. Considering attachment of the protein cysteine to carbohydrate on the antibody. I notice you have reagents that can couple to cysteine via maleimide, do you have a reagent that oxidizes carbohydrate groups and also a linker that couples to the resultant aldehydes?

We cannot recommend the scheme proposed. The way we typically conjugate through the carbohydrate groups is by oxidation, which would give one less control, not more, and the oxidative product is an aliphatic aldehyde, which is not as stable as the aromatic aldehyde. We get very controllable, reproducible results with our UV- traceable chemistry that links through the amino groups.

Recently I bought your crosslinking products HyNic and 4FB. I´m trying to crosslink peptide (3,000 KDa) with protein (50,000 KDa). To modify theses I use S-HyNic for the protein and S-4FB for peptide. When I mix the peptide with S-4FB ( 1 µg of S4FB/20 µl of DMSO), I always see turbidity and a precipitate. Do you know why this happens?

Peptides can be particularly difficult to modify due to their propensity for low aqueous solubility. Depending on the sequence, the peptide may only be soluble at high or low pH (due to acidic or basic residues.) Additionally, some residues such as A, F, L, V, are very hydrophobic at any pH. Since the S-4FB will modify all lysines present (as well as the N-terminus), it may be possible that the peptide is being over-modified with 4FB. Thermo Scientific offers some tips for working with troublesome sequences. Ultimately, it may be necessary to either optimize the sequence by substituting hydrophilic residues or by putting the HyNic on the peptide during SPPS. You can also change the strategy by putting the more hydrophilic HyNic on the peptide and the 4FB on the protein – but beware – it is easy to over-modify proteins with 4FB and they, too, will precipitate. I would shoot for 2-3 4FB molecules per mol of 50 KDa protein (ie., add 5 equivalents to the crude reaction with the protein at ~3 mg/mL.)

We are using your protein-oligo kit to attach peptides to oligos. My peptide precipitates out of solution once labeled with HyNic. What can I do to prevent this?

You should dissolve your peptide in DMF. Many peptides are difficult to work with because of solubility issues. Perform the modification reaction with 1 mole equivalent of HyNic, and the reaction should be performed in DMF with 2 mole equivalents of TEA (triethylamine). The peptide should be modified, and stay in solution. The 4FB-oligo should be dissolved in a 50:50 mix of conjugation buffer and DMF. Perform the conjugation reaction in this mixture.

Do you offer any amine-to-sulfhydryl crosslinkers with polyethylene glycol (PEG) spacer arms?

We have 2 crosslinkers that can react with thiol groups. MHPH (Cat. No. S-1009) which is maleimido HyNic, and MTFB (Cat. No. S-1035) which is maleimido 4FB. Our unique system is that HyNic reacts with 4FB to form a stable hydrazone bond. We have a long-chain crosslinker (PEG4/PFB) (Cat. No. S-1034) which reacts with amine groups and attaches 4FB.

We need to prepare 1:1 antibody:protein conjugates using your protein-protein crosslinking kit. Suggestions?

Example – obtaining antibody:30 kDa protein 1:1 conjugates. We suggest modifying the 30 kDa protein with HyNic and the antibody with 4FB. For HyNic, use 15–20X mole equivalents to have 2–3 per protein. For 4FB, use 10–20X mole equivalents to have 3–5 per antibody. Make a 1 mg/mL solution of each for modification. After a 2 hr modification, pass 130 µL, corresponding to 130 µg over the desalting column – should get about 100 µg of modified protein. With these 2 tubes, make some combinations for your conjugates (incubating 2 hr with 10X TurboLink buffer (Cat. No. S-2006-105)) and run the products on a gel to see which ratios give the best 1:1 conjugate. Scale up your conjugations after getting results from the gel.

Gel lanes:
Protein alone
Antibody alone
Pair-wise combinations – can set this up, using both modification conditions for both your antibody and protein:
0.5 / 1
1 / 1
1 / 0.5

For quenching the conjugation reaction, add a 50-fold molar excess of sulfobenzaldehyde to the reaction to react with any excess HyNic groups.

What is your silica bead modification protocol?

1) Weigh out 100 mg of silica beads.
2) Wash the silica beads 3 times with EtOH; pellet the bead on a centrifuge for 2 min at 750 x g, remove the supernatant. Bring the beads up in EtOH and repeat.
3) Make a 50 mM solution of Hynic silane (10 mg) in EtOH (500 µL), add 2% (10 µL) water to the solution to dissolve any remaining solids. This may require intensive vortexing to get the silane into solution.
4) Add the Hynic silane solution to the washed bead pellet such that the silane/silica ratio is 20% w/v (500 µL).
5) Vortex the bead sample and incubate at room temperature on a rotator for 30 min.
Note: Check the pH periodically with pH paper and be sure that it doesn’t go below pH 7.4 during the incubation steps!! Bring the pH to above 7.4 with 1 M NaOH if the pH drops.
6) Add an additional 2% (10 µL) water to the bead solution and continue the incubation for 15 min.
7) Add an additional 10%(50 µL) water to the bead solution and continue the incubation for 5 min.
8) The washing step is very important: Washed the beads 3X each with water, ethanol, water, PBS and Conjugation Buffer (100mM sodium phosphate, 150 mM NaCl, pH 6.0) in that order, using the spin protocol from step 1.
9) Check the supernatant to see if there is a significant A280 from the Hynic-silane; add 100 µL of supernatant to 900 µL of Buffer. The A280 should give a reading no higher than 0.05.
10) Bring the beads up in Conjugation Buffer such that the solution is a 20% w/v beads in Conjugation Buffer.

The Hynic-modified beads are now ready to be conjugated the 4FB-modified biomolecule. For large biomolecules (proteins and antibodies), 20 µg of protein/mg of bead is recommended for maximum conjugation. Allow the beads to conjugate with the protein overnight at room temperature on a rotor or shaker.

Does your chemistry work with Quantum Dots?

Our chemistry works very well with Qdot® Nanocrystals. If you are using amino dots to link peptides, I would recommend using sulfo S-4FB (S-1008) to modify the “Dots” and then use Hynic to modify your peptide. Our linking chemistry links through the ?-amino group on the lysine (S-Hynic, S-1002) or through a thiol group on a cysteine (MHPH, S-1009) allowing for direct modification of your peptide after synthesis. We also have a peptide reagent that adds our S-Hynic linker onto either end of the peptide during synthesis (6 BOC HNA, Cat. No. S-3003). For conjugation of carbohydrates, we recommend modifying your amino dots with S-Hynic (S-1002) and then oxidizing the carbohydrates with 10 mM sodium periodate and reacting the two modified bioconjugates.If you have carboxy-modified Quantum Dots, you will need to activate them with sulfo-NHS/EDC then quench with 100 mM EDA, 100 mM borate, pH 8.0 to amino-modify them prior to using any of our proprietary linkers.

How does the PEG4/PFB react with amines?

The product S-1034 (PEG4/PFB Long Chain Crosslinker) does react with primary amine groups and does have a PEG4 linker arm. When looking at the structure on the product data sheet, you notice the penta-fluoro phenyl group. This is the leaving group, and the mechanism is very similar to the NHS ester mechanism. The nitrogen of the amine group attacks the carbonyl carbon adjacent to this potential leaving group, and with some rearrangement, along with participation of water, the phenyl group leaves.

What are the advantages of using a quantifiable linker?

With the TriLink linkers (4FB and HyNic), you can easily quantitate the number of linkers you add to each biomolecule. As with labeling, for maximum sensitivity and reproducibility, it is important to avoid too many linkers, which leads to reduced immune reactivity, or too few linkers, which leads to weak signal or unconjugated material. Quantitative linkers allow for more control and reproducible steps during the conjugation reaction.

Should I add 4FB or HyNic to my amino-modified oligo?

We have found that our Hynic oligos have a tendency to dimerize if not used immediately after modification. If you are planning on immobilizing the oligo on 4FB beads, if the Hynic oligo is added to the 4FB magnetic microspheres immediately after desalting, then you shouldn’t see too much dimerization. We have used this scheme before with success. For these reasons, for typical reactions, such as antibody-oligo conjugations, you want to modify your oligo with 4FB, not with HyNic.

I want to conjugate protein A (43 kDa) to antibody IgG (150 kDa). We have both cross linkers (C6-SANH (HyNic) linker and C6-S-4FB linker). My protein is very sensitive to pH, buffer composition, temperature, and solvent. It’s very risky to incubate at room temperature for a long time. How many fold excess of linker would be suitable for this protein? Is it ok to incubate this reaction at 4 degrees (C) for 2 hr for modification? Please suggest the antibody IgG (150 kDa) amount. I will use C6-S-4FB crosslinker for the modification of antibody. For the conjugation reaction, which molar ratio would be suitable for best conjugation of both modified proteins (I am not sure it depends upon the amount of modified protein or crosslinker attached in modifying protein). The molecular weight of my protein A (43 kDa) is approx three times lesser than the antibody IgG (150 kDa). Is it ok to incubate the conjugation reaction for 12 hr at 4 degrees (C) with pH near 7 for best conjugation?

All of our modification data is at room temperature. While modification at 4 degrees may work, we cannot guarantee success. For the modification, we would suggest 15 equiv at 2 mg/mL protein A concentration. We suggest 10 equiv at 2 mg/mL. For the conjugations, we recommend a 1/1 mol/mol ratio. However, we suggest trying 1/1.5 and 1/2 antibody/protein A ratios on an initial small scale (~10 µg) reactions that can be analyzed by PAGE. Use 10X TurboLink buffer in the solution to optimize the conjugation reaction.

I am trying to conjugate an antibody to another protein with S-HyNic and S-4FB. In Table 1, MSR of S-HyNic saturates to 8 or 9. MSR of S-4FB saturates to 26 or 28. What makes the difference between those two values? Does S-HyNic or S-4FB or conjugate-product have any charges that affect the conjugation efficiency?

Different linkers have varying modification efficiencies. 4FB modifies much more efficiently than does HyNic. 4FB is very hydrophobic, and binds nonspecifically, initially, to protein hydrophobic regions, then is in close proximity to, and reacts with Lys residues very efficiently. HyNic does have a protonated group (pyridine), and does not crosslink to the biomolecule with the same efficiency as 4FB. The charge on the HyNic perhaps contributes to it’s lower modification efficiency, but does not affect its conjugation efficiency to 4FB.

We want to conjugate 10 mg of each of 2 proteins (lyophilized). One is 14 kDa and the other is 44 kDa.

You should obtain the larger Zeba columns from Pierce, if you want to perform a larger scale reaction. We would suggest performing the conjugation on a much smaller scale. Not more than 100 µg of protein. In that case, the smaller desalting columns would be sufficiently large. On the bottom of page 7 of the Protein-Protein conjugation kit manual, you are directed to add a large amount of HyNic in DMF because the amount of protein is large. Would suggest making 5 mg/mL stock solutions. You should scale this reaction down significantly, modifying just 100 µg of each protein. Would suggest modifying the larger protein with 15 equivalents of HyNic and the smaller protein with 5 equivalents of 4FB. 4FB is a much more efficient crosslinker.

I bought a protein-oligo conjugation kit from your company and I found that the recommended length of oligo is up to 100 base bairs. However, my oligo is 200 base pairs in length. Will the 200 bp oligo work in this kit?

The protein-oligo conjugation kit is to be used with an HPLC-purified oligo that has been amine modified. If you are doing an amine conjugation to your 200 bp oligo, the purification is crucial. You will need a significant number of nmol of oligo to perform a successful conjugation. You can work with our kit and you might have good success. However, as this strategy is outside our testing conditions, we cannot guarantee that you will have success with this product.

Has your company ever perform peptide-hapten conjugations for regulatory studies? Is there any method to analyze the peptide-hapten stability? This is important since if there is not a positive response after the immunization we should know whether the conjugation was stable or not. Is there any method to determine the amount of peptide that is conjugated to the hapten? Does your company provide a Quality Control (QC) for the conjugation procedure?

Several clients have had TriLink link small molecules, peptides, and oligonucleotides to KLH using TriLink’s bioconjugation technology, and in all cases, have isolated antibodies. Yes, the linkage between the peptide and the KLH is chromophoric and can be quantified spectrophotometrically. We can provide whatever data you need, including protein concentration and number of peptides/KLH.

Regarding conjugation of a HyNic peptide and 4FB oligo… we are mostly concerned with getting all of our 4FB sites on our DNA conjugated to protein, not with the efficiency of protein conjugating to the oligo. The instructions for conjugation seem to suggest a ~2:1 ratio of DNA to protein to obtain 95% protein conjugation. But, if we are more interested in the DNA, then I assume our ratio should be less. I am wondering how much less? Also, if we would like to store some of the HyNic peptide for later use, is it stable if stored in buffer at –80ºC?

If your main goal is to conjugate all of the 4FB oligo, then you may want to use a ratio closer to 1:1. The number of oligos a given protein can carry is largely governed by its surface area, which is roughly proportionate to its molecular weight. In our experience, an antibody has no difficulty conjugating to multiple oligos in the 20–30 nucleotide range. Smaller proteins would do better with a 1:1 or perhaps 2:1 DNA:protein ratio. Particularly, if you don’t mind having free protein in the crude reaction, then this is not a problem. I would add our 10X TurboLink catalyst buffer to drive the reaction to completion. Regarding the 4FB on the oligonucleotides, we order the amino-derivitized oligo and then incorporate the 4FB post-synthetically using our 4FB HNS linker. This process is quantitative in our experience. The ultimate purity of the oligo (in terms of full-length 4FB-modified) is therefore dependent on the incorporation efficiency of the amino linker. Therefore it is important to remember that although you might be adding 2 molar equivalents of oligo to your protein for conjugation, you’re really adding only 1.1 equivalent of 4FB oligo. The unmodified oligo cannot conjugate and will remain in the conjugation reaction. Regarding the peptide, it is most stable as a lyophilizate. If you have an analytical balance you can weigh out 1–2 mg to dissolve for your studies and store the rest lyophilized at –80ºC. Barring that possibility, you may dissolve the whole tube and aliquot it out to usable portions and store at –80 ºC. Avoid freeze-thaw of the peptide as this will cause it to precipitate and may affect the HyNic group adversely.

I am interested in your crosslinking products, and would like to know if you have any conjugation kits that have been tested successfully with protein-lipid or protein-carbohydrate combinations?

We have successfully used our technology to link proteins to lipids and carbohydrates. The lipid or carbohydrate in question has to be modified with an amine however, and the chemistry to do this isn’t trivial. If you have a lipid or carbohydrate which is already amine modified, then one of our kits will make the conjugation quite easy to perform. If you do not have amine-modified biomolecules, this sort of conjugation is often times performed by our custom conjugation group. We have done this before, and he can give you tips, suggestions, or a quote for a service to do this for you.

Does TriLink use SMCC (Pierce) to conjugate a peptides to proteins?

We do not use a single bi-functional linker like SMCC. Our linking chemistry links through the e-amino group on the lysine, or through a thiol group on a cysteine allowing for direct modification of your peptide after synthesis. We also have a peptide reagent that adds our S-Hynic linker onto either end of the peptide during synthesis. Our chemistry then requires a separate modification of the antibody/protein with a traceable linker that corresponds to S-Hynic. This linker is S-4FB. The resulting conjugate that forms when the two linkers come together has a hydrazone chromaphore that allows the end-user to know exactly how many peptides are on the antibody. This chemistry provides several advantages when compared to SMCC; namely, it is more reproducible, it is traceable, and it reacts specifically, preventing undesirable polymer formation.

If my TAT-HyNic peptide is dissolved in conjugation buffer; should I store at –20ºC? How long the does the solution stay active? What molar excess of TAT-HyNic should I use for antibody conjugation? After conjugation, can purification be done by using a desalting column or by dialysis? What kind of buffer is best for antibody-TAT storage?

Once in solution, the peptide should either be used ASAP or frozen at –80 ºC. Be sure to aliquot the peptide solution into usable portions prior to freezing to avoid the need to freeze-thaw. TAT-HyNic peptide conjugation. A 3–5 mole excess of TAT-HyNic peptide should be used. Purification can be performed with a diafiltration column with a 5 kDa MWCO or by dialysis. Storage buffer should be PBS with 0.5% azide.

How stable are HyNic and 4FB-modified proteins (stored in aqueous solution)?

4FB-modified proteins are stable at 4oC for up to one year. HyNic-modified proteins are not stable and should be used the same day they are modified, preferably. HyNic-modified proteins can be stored at –80?C in small, single-use aliquots, in conjugation buffer. No need to add glycerol. And, do not re-freeze after used – reason for the aliquots. Should be good for several months. You want the proteins frozen, not moving around in solution, with possible HyNic-amino group interaction.