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While adsorption of biomolecules to our passive surfaces is more electrostatic in nature, our CovaLink and Immobilizer Amino surfaces present an option for true covalent bonding of molecules with specific free residues. CovaLink possesses a secondary amine structure, which will covalently bond to biomolecules with a free carboxyl or phosphate group. This allows for a much more robust and specific coating, which will often tolerate more rigorous washing steps and eliminate the need for blocking.

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AminoLink and AminoLink Plus Supports are activated with aldehyde groups which will react with primary amines to form Schiff bases, which are reduced to stable, non-reversible secondary amines. Coupling efficiency often exceeds 85% with this support.

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Both react with primary amines, but AminoLink Coupling Resin is a two-step conjugation. First a Schiff base is formed between the amine and the aldehyde on the resin, which is then reduced to a stable secondary amine with sodium cyanoborohydride, whereas NHS-Activated Agarose utilizes an NHS ester to form an amide bond in a one-step conjugation.

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NHS- activated agarose reacts with primary and secondary amines to form stable amide and imide linkages respectively. Most proteins can be coupled in 30 mins with greater than 85% coupling efficiency. No hazardous chemicals are used, and the agarose is adaptable to use with spin columns, batch methods, or FPLC. The resin is resusable typically up to 10 purifications with the immobilized ligand, and has a high binding capacity (>30 mg/mL slurry or >25 mg/mL dry resin).

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Here are possible causes and solutions:

- The kit has expired or has been stored incorrectly: When properly stored, the components of the CBQCA Protein Quantitation Kit should be stable for at least 6 months. Upon receipt, the kit should be stored at -20 degrees C and protected from light. Solutions of Components A, C, and D may be stored at 4 degress C for several days or at -20 degrees C for long-term storage.
- Poor excitation/emission filter settings: Read the samples in a fluorescence microplate reader, standard fluorometer or minifluorometer using excitation/emission wavelengths of approximately 465/550 nm.
- pH is too high or too low: Dilute standards and samples in 100 mM sodium borate buffer, pH 9.3. Other buffers can be used, but labeling is optimal near pH 9.3. Prepare standards and samples under the same conditions to account for buffer pH effects.
- Sample buffer contains primary amines or ammonium salts: The CBQCA reagent reacts with glutamine, asparagine and primary amines, such as the epsilon amine on lysine. It does not react with histidine or secondary amines. Therefore the sample should be free of ammonium salts and contaminating amines such as Tris or glycine.
- Sample buffer contains high concentrations of thiols: PThiols should not exceed 100µM final concentration in the assay. Add a higher concentration of N-ethylmaleimide (NEM) to block thiols.
- Sample buffer contains other components that are affecting the assay: The CBQCA assay is generally tolerant of the presence of lipids, detergents, glycerol, sucrose, salts and sodium azide, but these contaminants do affect the signal and sensitivity. Prepare standards and samples under the same conditions to account for buffer composition effects.

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The following list offers a brief description of the features of Nunc products and their specific applications:
- Nunc-Immuno Plates, MaxiSorp surface - These plates are designed for solid phase immuno assays and have a polystyrene surface with high affinity for polar groups and hydrophilic molecules. These 96-well plates are available with flat (F), round (U), or (C) bottom-well designs.
- Nunc-Immuno Plates, PolySorp surface - These plates have a polystyrene surface which adsorps less polar molecules compared to the MaxiSorp surface and has a high affinity for hydrophobic groups. These 96-well plates are available with flat (F), round (U), or (C) bottom-well designs.
- Nunc-Immuno Modules - These modules are designed for solid-phase immuno assays. The modules are available in 8-, 12- or 16-well formats with (F), (U), and (C) bottom-well and 8-well BreakApart with (C) bottom-well designs. These different formats allow one to choose the style which is appropriate for their assay design.
BreakApart Modules and LockWell Modules can be used for radioimmunoassays.
- Nunc StarWell Immuno Breakable Modules - The 8-well modules feature eight fins on the inner wall of the (C) bottom wells. This design increases the surface area by 50%. The increase in surface area allows more molecules to be immobilized, increasing assay signal. The fin configuration provides shorter diffusion distance to the surface, reducing incubation times.
- LockWell Immuno Breakable Modules - Each plate consists of 1 x 8 breakable strips. These strips are assembled in a designed frame which locks each well into place by a spring lock. This spring lock design orientates each well at the same horizontal level allowing uniform washing and reading. The LockWell Modules are available with round (U), (C) or StarWell bottom-well designs.
- Plates and Modules with (CovaLink) Covalent Binding Surfaces: These plates can be used to bind proteins, peptides, DNA or carbohydrates. Covalent linkage occurs via secondary amine group. This allows a specific orientation and provides improved stability compared to passive adsorption due to chemical binding between molecules. Binding of molecules on PolySorp and MaxiSorp surfaces is obtained by passive adsorption. CovaLink is a surface grafted with secondary amino groups (<NH) which serves as a bridge for covalent coupling. The covalent binding can immobilize small molecules such as biotin or peptides which may otherwise bind weakly by physical adsorption and facilitate the recognition by detection molecule.
- Nunc FluoroNunc/LumiNunc 96-Well Plates: These plates are optimized for IFMA (Immunofluorometric Assays) or FIA (Fluorometric Immuno Assays). The transparent polystyrene plates give a low background fluorescence and are optimal where a read-through system is used. The white plates/modules provide maximum reflection of fluorescence signal while maintaining low background. The white plates/modules are often used for epifluorescence reading. Black modules reduce background fluorescence and minimize back-scatter light which is often encountered in epifluorescence.

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Sulfo-SBED allows one to transfer a biotin tag from an amine-containing protein to a second protein (reacted with the phenyl azide). The amine-containing protein can be removed by reduction of the disulfide in the Sulfo-SBED spacer arm. This leaves just the second biotinylated protein for detection.

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These types of spots are caused by some component of the IEF sample buffer that runs into the gel during the second dimension separation and is stained by SYPRO Ruby Protein Gel Stain. SYPRO Ruby dye is attracted to amines, such as amines in lysine, arginine and histidine containing peptides, but also amines in detergents and ampholytes. The simplest solution is to try a different IEF buffer formulation that does not cause this artifact. Possibly, a thorough overnight fixation in several changes of 50% methanol/7% acetic acid will wash out the contaminant.

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The ARES Alexa Fluor DNA Labeling Kits provides a versatile, two-step method for labeling DNA with our Alexa Fluor dyes. In the first step, an amine-modified nucleotide is incorporated into DNA using enzymatic labeling methods. In the second step, the amine-modified DNA is chemically labeled using our amine-reactive Alexa Fluor dyes. The labeled probes can be used for:

- Fluorescence in situ hybridization (FISH). See Buster DW, Daniel SG, Nguyen HQ, et al. (2013) SCFSlimb ubiquitin ligase suppresses condensin II–mediated nuclear reorganization by degrading Cap-H2, J Cell Biol 201(1):49-63.
- Microarray techniques. See Shin HH, Hwang BH, Seo JH et al. (2014) Specific discrimination of three pathogenic Salmonella enterica subsp. enterica serotypes by carB-based oligonucleotide microarray. Appl Environ Microbiol 80(1):366-373.

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Crosslinkers can be classified as homobifunctional or heterobifunctional: -Homobifunctional crosslinkers have identical reactive groups at either end of a spacer arm, and generally they must be used in one-step reaction procedures to randomly "fix" or polymerize molecules containing like functional groups. For example, adding an amine-to-amine crosslinker to a cell lysate will result in random conjugation of protein subunits, interacting proteins and any other polypeptides whose lysine side chains happen to be near each other in the solution. This is ideal for capturing a "snapshot" of all protein interactions but cannot provide the precision needed for other types of crosslinking applications. -Heterobifunctional crosslinkers possess different reactive groups at either end. These reagents not only allow for single-step conjugation of molecules that have the respective target functional groups, but they also allow for sequential (two-step) conjugations that minimize undesirable polymerization or self-conjugation. In sequential procedures, heterobifunctional reagents are reacted with one protein using the most labile group of the crosslinker first. After removing excess non-reacted crosslinker, the modified first protein is added to a solution containing the second protein where reaction through the second reactive group of the crosslinker occurs.

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Yes. Elution of isolated proteins without release of the specific antibodies, and reuse of the immobilized antibodies requires covalent crosslinking. Secondary antibodies are supplied covalently bound to the beads. Crosslinking the bound primary antibodies to the secondary antibody-coated Dynabeads magnetic beads will covalently bind the primary antibodies to the secondary antibodies. A protocol for crosslinking is available in Dynabeads magnetic beads product manuals. However, we cannot guarantee the full recovery of your antibody activity, as this varies from antibody to antibody. Commercially available crosslinkers reacting with protein amine groups can be used. For example, see the crosslinker, BS3 (Cat. No. A39266). Optimization of the cross-linking reaction may be required.

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