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Hydrogen bromide agarose gel

NegotiableUpdate on 05/06

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Nature of the Manufacturer: Producers

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Overview

CNBr activated Sepharose 4B $r $n Hydrogen bromide agarose gel 4B $r $n Application: affinity chromatography filler $r $n Package: 15g

Product Details

CNBr activated Sepharose 4B

Hydrogen bromide agarose gel4B

Application: Affinity chromatography packing material

Packaging: 15g, 100g, 250g solid packaging

To ensure the performance and worry free operation of the product, please read this manual carefully before use. If you have any questions, please consult our company's after-sales technical support or local sales personnel (see appendix).

1. Product Introduction

CNBr Focusose 4FF is a fast flow purification medium containing cyanate ester groups activated by cyanogen bromide, suitable for coupling amino containing biomolecules such as proteins, peptides, and nucleic acids. After repeated validation in the purification process of biomedicine, it has been widely applied.

The characteristics are as follows:

a. Widely used,Hydrogen bromide agarose gelSuitable for coupling amino containing biomolecules.

b. Multi point coupling, simple, flexible, fast, and effective, can efficiently maintain the biological activity and stability of biomolecules.

c. Fast flow rate, high yield, and easy to scale up.

Table 1: Performance Parameters of Media

matrix	Highly cross-linked 4% agarose
particle size range	45-165µm
average particle size	90µm
Combined with load capacity	30mg (Trypsinogen)/ml (medium)
ph stability	3-11 (Long term) 2-11 (Short term)
maximum flow rate	700cm/h
Operating pressure	≤0.3MPa
stock solution	100% acetone
Storage temperature	4-8℃

2. Coupling conditions

a. (Liquid A) Cleaning

Take an appropriate amount of settling gel (0.83g, approximately 1.0ml after cleaning), resuspend the medium with 5 times the volume of solution A, and drain the solution after 5 minutes. Repeat this step 5 times.

Note: This step is used to activate the medium, ensuring that the cleaning volume of solution A is sufficient and the cleaning time is fixed at around 0.5 hours (if the time is too long, the functional groups on the medium will hydrolyze).

b. Preparation of ligand solution

Dissolve the biomolecules to be coupled in solution B or replace them with solution B (biomolecule concentration is 1-10mg/ml, recommended 5mg/ml).

Note: Ensure that the pH and salt concentration during coupling are consistent with solution B.

c. Coupling

Mix the cleaned medium and prepared sample in equal proportions (volume ratio), gently mix at room temperature for 3-4 hours. After confirming successful coupling (coupling efficiency is determined by measuring the content of biomolecules before and after coupling), drain the solution.

Note: It is recommended to couple at room temperature for 3-4 hours. For unstable ligands, it is recommended to couple at 4-8 ℃ overnight.

d. (C liquid) cleaning

Resuspend the coupled medium with 5 times the volume of C solution, and then drain the solution. Repeat this step three more times.

Note: This step is used to clean residual biomolecules in the medium, and cleaning should be done with *.

e. Closed off

Resuspend with 5 times the volume of C solution, gently mix at room temperature for 3-4 hours, and then drain the solution.

Note: This step is used to seal the functional groups on the medium. Mix gently at room temperature for 3-4 hours.

f. (D liquid and E liquid) cleaning

Resuspend the sealed medium with 5 times the volume of D solution, and drain the solution after 5 minutes; Resuspend the medium with 5 times the volume of E solution, and drain the solution after 5 minutes. Repeat this step 3 times.

Note: This step is used for acid-base cleaning to remove biomolecules that are not tightly coupled.

g. Save

Resuspend the medium with 5 times the volume of purified water and drain it. Then resuspend the medium with 5 times the volume of 20% ethanol and drain it. Finally, soak it in 20% ethanol for preservation.

Note: This step is used to preserve the medium and prevent microbial growth.

h. Solution preparation

A solution: 0.001MHCl, 0.5M NaCl, adjusted pH 3.0, stored at 4-8 ℃ (A solution needs to be pre cooled before use).

B solution: 0.2M NaHCO3, 0.5 M NaCl, adjust pH 8.3 (if the biomolecule to be coupled is IgG, pH=8.5-9.0）， Store at room temperature.

C solution: 0.1M Tris HCl, adjusted to pH 8.3, stored at room temperature.

Solution D: 0.05M Tris HCl, 0.5M NaCl, adjusted to pH 8.5, stored at room temperature.

E solution: 0.05M GAN amino acid, 0.5M NaCl, adjusted pH 3.5, stored at room temperature.

F solution: 1.0M NaCl, stored at room temperature.

3. Cleaning

After cleaning, some strongly binding substances (such as strongly bound proteins, denatured proteins, lipids, etc.) can be removed to achieve excellent performance of the recovery medium (such as loading capacity, fluidity, column efficiency, etc.).

It is recommended to clean every 5 uses, and the specific cleaning frequency should be adjusted according to the cleanliness of the purified initial sample.

⑴ Conventional cleaning

a. Rinse with 5-10 times the volume of purified water.

b. Rinse with 5-10 times the column volume of D solution.

c. Rinse with 5-10 times the column volume of E solution.

d. Rinse with 5-10 times the column volume of F solution.

e. Rinse with 5-10 times the volume of purified water.

f. Rinse with 5-10 times the column volume of 20% ethanol and store.

⑵ Intense cleaning

a. After rinsing with 2-5 times the column volume of 0.2% non-ionic detergent, immediately rinse with 5-10 times the column volume of purified water.

b. After rinsing with 2-5 times the column volume of 6M guanidine hydrochloride, immediately rinse with 5-10 times the column volume of purified water.

c. Rinse with 5-10 times the column volume of 20% ethanol and store.

Note: The applicability of severe cleaning conditions mainly depends on the stability of the coupled biomolecules. Before using these conditions for cleaning, it is recommended to conduct a small-scale pre experiment to verify the stability of the biomolecules.

4. Application Cases

Case One

Case Two

5. Common problems

Table 2: Common Problems and Solutions

problem	Possible reasons	solution
Low coupling efficiency	1. The salt concentration or pH of solution B is incorrect	Check if the preparation of solution B is correct
	2. Insufficient coupling time	Extend coupling time
	3. Inappropriate pre activated filler	Try other types of pre activated fillers
The target substance does not bind to the medium during purification Or the binding amount is relatively low	1. Sample overload	Reduce sample size
	2. The sample loading speed is too fast	Reduce the sample flow rate
	3. Protein or lipid aggregation in the medium	Timely and effectively clean the medium
	4. Sample deactivation during storage or loading process	Properly store the sample to be purified to maintain its activity
	5. The binding ratio between ligands and target compounds is relatively low	Attempt to increase the concentration of ligands during coupling
	6. Degradation of ligands during coupling or cleaning processes	Detecting the stability of ligands during coupling or cleaning
No target substance was collected during elution Or only collect a small amount of target objects	1. The target substance is not bound to the medium or has a small binding amount	Reduce the sample flow rate and check the binding ability of the medium
	2. Inappropriate elution conditions	Change the corresponding elution conditions or enhance the elution ability of the eluent
	3. The target substance appears to aggregate and precipitate under eluent conditions	Detecting the stability of the target substance under eluent conditions (such as salt concentration and pH)
The purity of the target substance is relatively low	1. The sample has not undergone pre-treatment	The sample must be centrifuged or filtered before being loaded onto the column
	2. Sample viscosity is too high	Dilute the sample appropriately with equilibrium solution to reduce viscosity.
	3. Do not wash impurities*	Increase the washing volume until the baseline is stable and consistent with the equilibrium solution
	4. Impurity proteins or lipids aggregate and precipitate in the medium	Timely and effectively clean the medium
	5. Poor elution conditions, too fast elution speed, and too steep gradient.	Adjust elution conditions
	6. Degradation of the target substance occurs	Detecting the stability of the target object
	7. Poor filling effect of column materials	Reload or purchase
	8. Impurities exhibit non-specific adsorption	Proper selection of additives to reduce non-specific adsorption
	9. There is a large sample storage volume at the top of the separation column	Reinstall the column or reduce the storage volume of the sample
	10. Microbial growth in the medium	After using the medium, please store it correctly and promptly
Decreased medium load	1. The sample loading speed is too fast	Reduce the sample flow rate
	2. Proteins or lipids aggregate in the medium, leading to a decrease in load.	Timely clean the medium
	3. Excessive use leads to ligand detachment	Reconnect with a new medium
	4. The sample becomes inactive during storage or loading, and cannot bind well with the ligand	Properly store the sample to be purified to maintain its activity
Slow rise of chromatographic peak	The medium is packed too tightly	Reinstall the column
Chromatographic peak tailing	Medium loading too loose	Reinstall the column
There are cracks or dryness in the column bed	Leakage or introduction of large volume bubbles	Check for leaks or bubbles in the pipeline and reinstall the column
Slow liquid flow	1. Protein or lipid aggregation	Timely clean the medium or filter membrane
	2. Protein precipitation in the medium	Adjust the components of the equilibrium solution and eluent to maintain the stability of the target substance and the binding efficiency of the medium
	3. Microbial growth in the separation column	The reagents used must be filtered and degassed; The sample must be centrifuged or filtered before being loaded onto the column

6. Ordering Information

Table 3: Ordering Information Table

product	Specification (ml)	Item Number
CNBr Focurose 4FF	25	HZ1001-2
CNBr Focurose 4FF	100	HZ1001-2
CNBr Focurose 4FF	500	HZ1001-2
CNBr Focurose 4FF	1000	HZ1001-2

Note: For purchasing large-sized packaged products or other products, please consult our local sales or pre-sales technical support.