We routinely quantify the biotin-binding capacity of our beads using fluorescein-biotin. However, since it is very difficult to ascertain the effect of the bead on quantum yield and other properties of the fluor, we avoid this situation by fluorescently quantifying the amount of unbound vs. bound bioin-fluorophore left in solution. By simple subtraction, we obtain the amount bound. If you use this technique, it is always wise to run a control bead without bound streptavidin if possible as well as a no bead control.
The NanoLink™ 4FB beads are used for direct binding of HyNic-modified biomolecules. To learn more, please visit the NanoLink™ 4FB product page.
We haven’t performed any specific tests, but streptavidin is a pretty stable protein, so concentrations of these reagents that are “protein-friendly” would be fine with regards to the streptavidin-coated beads.
Not specifically, but we do recommend using a blocking agent such as casein. We sell a bead block for this purpose.
The beads are stable over a wide pH range, from pH 3 to 9.
As with all streptavidin beads, excessive heating will lead to dissociation of the streptavidin tetramer and leaching of monomeric subunits. We don’t recommend heating the streptavidin beads over 40C.
We have never used NP-40 with our streptavidin beads; nevertheless, this detergent should be completely compatible with our beads. The literature is full of references in which NP-40 at a final concentration of 0.1% is used to wash streptavidin beads prior to addition of biotinylated molecules. In this regard, our streptavidin beads should be no different and fully compatible with the use of this detergent. The covalent bond used to immobilize streptavidin on the beads is not affected by the presence of this detergent. The only detergent that we definitely know destroys all biotin binding capacity is the denaturing ionic detergent SDS.
For immobilization of proteins, we recommend using a casein buffer. We have a bead block buffer available (Cat. No. S-4023-025).Elution conditions:
We recommend using a glycine buffer (pH 2.8) for elution. You should collect the antigen in a neutral phosphate buffer, to limit exposure to the harsh pH conditions.Clumping Solutions:
To obtain a mono-disperse population of NanoLink™ Streptavidin beads, these procedures will help:
1. Vortex the beads for 1 minute in their original container to resuspend the pellet.
2. Remove a 10 µL aliquot (mass equivalent to 100 µg of beads) from the dispersed suspension using a P-10 pipette. Add the aliquot directly into a previously prepared solution of 1 M NaCl containing 1% Tween-20 and gently pipette up and down several times to disperse the beads while avoiding foam.
3. Immerse the tube containing the beads into a water bath sonicator (e.g., Branson 1200, 120 volts 50-60 Hz) for three min.
4. Remove a small aliquot of the beads and place them on a standard light microscope (e.g., 100X to 400X) to confirm monodispersity.
5. Once monodisperse, the beads can be washed twice (with a magnet) using molecular grade water. (Note: once the beads are monodisperse, they take a lot longer to pull them to the walls of the tube using a magnetic field (up to two full min).
6. Once the beads are monodisperse, they can be placed into any other suitable buffer you wish with or without Tween-20 and the majority of the population will remain monodisperse. Over time, (for example overnight), the beads will begin to reform aggregates due to the nature of streptavidin itself. This cannot be helped.
Important comments and notes: 1 M NaCl containing 1% Tween-20 combined with sonication does not adversely affect the biotin binding capacity of the NanoLink™ beads. Do not attempt to sonicate or disperse larger masses of beads at one time or in a larger volume. Attempts to disperse the entire volume of beads or even a smaller volume such as 100 µL beads (1 mg) at a single time is not effective. A large amount of sonication energy must be focused on a small volume and mass of beads (e.g., 100 µg in 100 µL) to fully re-disperse the beads into their mono-disperse state. Never use SDS at any concentration in an attempt to disperse the beads since this abolishes their biotin binding capacity.
We have sometimes noticed aggregation with NanoLink™ SA beads, but have recently (Nov 2010) modified our SOP to greatly reduce clumping issues. This can occur from the way the beads are prepared and the SA crosslinked. If you have a sonicator available, you might try performing a 1 minute sonication to disrupt aggregates. After a 30–60 sec sonication treatment, there was very little clumping observed, mostly single beads. Since the beads are very concentrated to begin with (10 mg/mL), the recommendation is that once diluted, that you sonicate your sample for 30–60 sec prior to your application. Beads obtained after November 2010 should experience very little clumping.
The 4FB magnetic microspheres are designed to work for Hynic-modified, small biomolecules (such as oligos and peptides). You can certainly use the 4FB magnetic microspheres in conjunction with the Hynic modified oligos or Hynic-modified peptides. The 4FB microspheres work fine at temperatures of 45?C. You can even go up to 60?C for up to five minutes and still have good stability. The Hynic oligo is not very stable due to polymerization, therefore we do not recommend that you use high temperatures on the Hynic modified oligo by itself, but once it is reacted with the 4FB, the bis aryl hydrazone oligo is very stable in temperatures up to 94?C.
The composition of our bead blocking solution is:
5 mg/mL Hammarsten-grade casein in 25 mM Tris, 150 mM NaCl, pH 7.4 containing 600 ppm Kathon anti-microbial agent and 0.05% anti-foam A.
You CAN use BSA in place of casein, but sometimes BSA may contain a small amount of biotin, which will bind to the SA. If BSA is not a problem with any of your downstream applications, then yes, it should be fine to use this as a blocking agent
Many researchers do not want blocked beads because the blocking protein can and does leach some. Not much but some. It is not covalently linked, so some leaching occurs. Some researchers do not want any protein leaching since this would interfere, for example, with protein sequencing and the specificity of the beads in their particular application. Other researchers require low nonspecific binding with very high binding capacity and don’t care about leaching. These researchers do want a blocked bead. If one is going to capture biotinylated DNA, you probably don’t need blocked beads. pH and detergents turn out to be more important factors for reducing DNA-based NSB than any other factors. If one is going to capture biotinylated antibody or proteins, one should block the beads for optimal results and minimal NSB. For some applications, pre-blocked beads would be a good thing, and for others, not needed. Regarding reduction of binding capacity using blocked beads…experiments we have done indicate that binding capacity is reduced for antibodies. But the reason for this is because 15% of the binding is NSB to begin with, and not related to biotin/streptavidin interactions. Even after extensive casein blocking, our NanoLink™ Streptavidin beads still bind 250 µg of biotinylated IgG vs. 80 µg for Invitrogen’s product (based on their website) per mg. So after blocking, we still bind 3X more antibody than anybody else. Without blocking, we capture a great deal more biotinylated IgG, but this increased capture is nonspecific binding. I believe that we have captured up to 400 µg of biotinylated IgG per mg of NanoLink™ without blocking the beads first. So yes, you will see a reduction in binding of biotinylated proteins, but this reduction is not due to loss of streptavidin, but rather blocking of NSB binding sites.
Depending on your application, Solulink recommends blocking streptavidin beads. If you are capturing non-phosphorylated proteins we recommend blocking with Bead Blocking Solution (Cat. No. S-4023-025). If you are capturing DNA or RNA, we recommend blocking with yeast RNA, salmon sperm, or herring sperm DNA at 10 mg/mL, if the presence of blocking DNA or RNA are not a factor in your downstream application.
We DO think that our NanoLink™ streptavidin-coated beads might provide you with lower nonspecific protein binding to your biotinylated biomolecule (compared to competing products) for 2 reasons. (1) Because of the highly crosslinked streptavidin (using our unique, patented chemistry) there is less polymer surface area on the bead exposed for proteins or nucleic acids to adhere to. This in itself should reduce NSB for you. Our extensively crossliked streptavidin is the reason the biotin binding capacity of these beads is so high. (2) And…because of the high biotin binding capacity, you can use FEWER beads in your assay…again reducing nonspecific protein binding.
All of the beads should be attached to antibody if you use a 10 to 20-fold molar excess of biotinylated product. Washing away unbound antibody is straightforward – you can magnetize the beads and wash them to remove unbound antibody. You can repeat this a few times, it is not trivial to remove any unbound beads from bound beads.
Random DNA sequences may bind to the surface of the magnetic beads, but only very weakly, if at all. The non-biotinylated sequences can easily be washed away from the beads. The crosslinked streptavidin doesn’t prevent binding.
We have 2 sizes of beads to fit the needs of specific applications. Some customers need a uniform sized bead. The MagnaLink™ beads offer this. For MOST applications however, this is not necessary. Some microarray applications might require a certain size as well. The larger bead will settle faster if that might be an issue in your application.
Higher binding capacity means greater capture using less bead mass. Less bead mass for your immobilization assays results in better assay capture and increased savings.
We currently offer two different types of magnetic beads; NanoLink™ is a less-uniform bead mixture which has 150 and 800 nm diameter beads, and MagnaLink™ is a very uniform bead with a 2.8 µm diameter. Both bead types are made with magnetite encapsulated by a non-styrene polymer surface. Unless you are particularly concerned with uniformity (e.g., for NMR, flow cytometry, etc.) the NanoLink™ beads are particularly suited for high-capacity capture of oligos, peptides, and antibodies. Both bead types come in three different varieties; streptavidin coated, 4FB linker coated, and amine coated. As a custom service, we can get other bead types for you, and link them to 4FB or streptavidin. And, we can add a linker with an extended spacer, but currently offer only streptavidin-coated, 4FB-coated, and amino beads as catalog products.
Bead size: bimodal (non-uniform) distribution (~150 nm to ~800 nm)
Density : 1.5 g/cm3
Magnetite content (Fe3O4): 40% (w/w)
Magnetic moment: 25 emu/g (electromagnetic units per gram)
Bead size; 2.8 micron (uniform distribution)
Density : 1.9 g/cm3
Magnetite content (Fe3O4): 50% (w/w)
Magnetic moment: N/A