Product name: NHS-Sepharose FF


English name: NHS Activated NUPharose Fast Flow, NHS NUPharose FF


Catalog Number: NRPB11L


Specifications: 1ml, 10 ml, 100 ml, 1L, special specifications customized


Transportation: 2 ~ 25℃, normal pressure, avoid light


Storage: 100% isopropanol, -20℃


Shelf life: 1 year






Related introduction:


NHS-Sepharose FF is a bio-affinity chromatography separation medium formed by NHS activation of agarose gel microspheres. The product retains the excellent hydrophilicity of agarose and the large grid structure, and has good compatibility with biologically active macromolecules. It has the characteristics of high capacity, low non-specific adsorption, and fast flow rate. The activated agarose gel can gently react with amino groups, and is particularly suitable for coupling with functional biological macromolecules such as antibodies, streptavidin, enzymes, and nucleic acids.






Technical index:


Exterior


Milky white translucent gelatinous gel


Matrix


4% agarose gel


Ligand


NHS


Ligand Density


≥15 μmol/mL


Protein load


≥10 mg/ml IgG


Back pressure resistant


0.3 MPa


Particle size range


60 ~ 180μm


Operating temperature


4 ~ 40℃


Matrix pH stability


3 ~ 12 (long time); 2 ~ 14 (short time)


Matrix chemical stability


Stable in the following solutions: commonly used aqueous buffer; 1 mol/L NaOH; 1 mol/L acetic acid (pH 4.0); 6 mol/L guanidine hydrochloride; 70% ethanol.






Applications:


Experiment name: NHS-Sepharose FF coupled antibody.


Experimental steps:


1. Prepare antibody: Dissolve 100 mg IgG with 10 ml coupling buffer (0.2 M NaHCO3, 0.5 M NaCl, pH 8.3), and reserve 0.1 ml to determine the coupling efficiency.


2. Coupling reaction: Take 10 ml of NHS-Sepharose FF, wash with 50 ml of 1 mM HCl three times to remove the preservation solution, add it to the antibody coupling solution immediately after washing, and shake for 6 hours or overnight at 4°C. Carry out the coupling reaction.


3. Blocking: filter the reaction solution, wash the coupled gel with 50 ml coupling buffer, then use 10 ml blocking buffer (0.2 M NaHCO3, 0.5 M NaCl, 0.5 M ethanolamine, pH 8.3), shake at room temperature Block the uncoupled group for 4 hours (or overnight), and wash it off with 20 ml of distilled water after blocking.


4. Detection: The reaction solution before and after the coupling was tested and compared with the coupling efficiency by SDS-PAGE. The coupled filler was used to determine the function and capacity according to the characteristics of the ligand.






Precautions:


1. If you dont know the stability of the ligand, you can choose different pH buffers and temperatures, use 0.5 ~ 1 ml of the solution with the corresponding concentration of the ligand, simulate the coupling conditions without adding fillers, and observe whether the solution will precipitate. If there is precipitation to produce the corresponding coupling effect, it is not good. You can slightly lower the pH or temperature. At the same time, you can add about 5% of glycerol or PEG to protect the protein to avoid precipitation. In short, it is best to understand the solubility and stability of any ligand. To avoid losses caused by experimental failure.


2. If the coupled ligand is unstable, shake the coupling on a shaker at 4°C for 1-12 hours to maintain the activity of the ligand. Clean the packing as soon as possible after coupling the ligand to avoid inactivation of the ligand under high pH conditions.


3. The more stable ligand can be directly dissolved in 0.2 M sodium bicarbonate, and the temperature can be appropriately increased. For example, at room temperature, the coupling time can be shortened to 1 to 2 hours.


4. The optimal pH for coupling protein is 7.5 ~ 9.0, at room temperature or 4°C. Increasing the temperature can increase the coupling efficiency of the ligand. Choose the appropriate pH and temperature according to the stability of the protein. The concentration of the coupling protein solution is preferably 5 ~ 15 mg/ml. The ratio of the volume of the gel to the volume of the ligand solution is preferably 1:0.5 ~ 1:2.0.


5. The coupling buffer can be carbonate, borate and phosphate buffers, but amino buffer systems, such as Tris-HCl buffer, must not be used.


6. The blocking buffer generally uses small molecule reagents with amino groups, such as ethanolamine, Tris, etc., and the pH can be completely blocked after a few hours at room temperature of 8-9. The recommended blocking buffer is 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3 or 0.1 M Tris-HCl, pH 8.5.


7. Try to use gentle mixing methods in coupling, such as shaking, turning, rotating, etc. If you use a magnetic stirrer or mechanical stirring, you need to reduce the stirring speed, as long as the filler can be mixed and completely suspended, so as to avoid violent stirring to break the gel microspheres.


8. NHS-Sepharose FF should be used now to avoid excessively long time and reduce the activity.

Warm tips: Suzhou Beike nano products are only used for scientific research, not for human body,different batches of products have different specifications and performance

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