BESTCHROM Diamond Blue Affinity Chromatography Resin Instruction Manual

Diamond Blue Affinity chromatography resin
Instruction for use![BESTCHROM Diamond Blue Affinity Chromatography Resin

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Introduction

Diamond Blue is a kind of affinity resin based on fine particles and high rigidity agarose. The ligand is Cibacron Blue 3GA, which is physically and chemically stable. Since the ligand is not easy to fall off, it can enjoy long service life and a wide range of applications. These mediators can not only bind to proteins through specific interactions,but also non-specific binding to proteins through electric charges.Diamond Blue has been widely used in the separation and purification of various proteins, such as dehydrogenase, kinase, transferase, serum albumin, interferon and plasma proteins. Compared to Blue Bestarose FF, Diamond Blue has better pressure flow rate performance and higher load capacity, helping to improve process efficiency and reduce cost.

Technical characteristics

40℃ 12h：1M NaOH, 70% acetic acid
Max. pressure| 0.5 MPa
Flow rate| 1200cm/h (0.5MPa BXK 100/500,H=20 cm 20℃)
pH stability| 2~13(working),2~13(CIP)
Temperature tolerance| 2~40 ℃ , Can’t freeze, Can tolerate 121 ℃  high pressure sterilization
Storage| 2~8℃，20% ethanol with 0.1M KH2PO4(pH 8.0)

+ Average particle size is the accumulated resin particle size of packing volume distribution
++ Residence time: 4min

Method of chromatographic

3.1 Column packing
Note：It is best to equilibrate the resin slurry to room temperature before column packing.

• According the column volume to calculate the amount of resin.
  Resin volume=column volume×1.15 (Compression factor=1.15)

According to the volume of the settlement resin required, the suspended slurry of the resin required is calculated by the follow:
Required resin slurry¹ concentration.The original concentration of resin slurry volume = Settlement resin volume ÷ Resin slurry¹ is shown in the follow table.original concentration of resin slurry¹ is shown in the follow table.

1: It refers to the original packaging resin slurry sold by Bestchrom.
Note: For non-original packaging, customer can calculate the required volume according to the actual concentration of resin slurry.

• Washing the resin: Suspend the resin by shaking and pour into a funnel, remove the liquid, and wash 3 times with about 3mL packing solution(20% ethanol + 0.4M NaCl) /mL resin. Use a glass stick or stirrer to stir each time you add the packing solution, in order to better clean the storage solution.

• Prepare the packing slurry: Move the washed resin from the funnel into a beaker or other appropriate container, and add packing solution to obtain a 50%~75% slurry concentration, stir well and set aside for use.

• Take a cleaned BXK column (BXK series columns with diameters ranging from 1cm to 30cm can satisfy different scale chromatography applications).Take BXK16/20 for example, purge the bubbles trapped at the end-piece net by draining some packing solution through the column outlet. Leave about 1cm water at the bottom of the column and close the bottom outlet. Adjust the column so that it is perpendicular to the ground.

• Slowly pour the slurry into the column at one time (use a packing reservoir if necessary). Do not bring any air bubbles into the column. Packing reservoir: Empty glasstube with same diameter as the BXK column.

• Fill the remainder of the column with packing solution.Connect the packing reservoir to the chromatography system, open the flow rate, drain the bubbles in the hose, close the flow rate, and tighten the top cover of the packing reservoir.

• After pouring, stir well again with Stirrer, and then wash the resin particles on the inner wall of the column from top to bottom with the packing solution, and let the resin settle naturally until there is about 1cm of clarifying solution on the suspension.Mount the adapter and connect the adapter to the chromatography system or peristaltic pump.Lower the adapter to descend to contact with the clarifying solution and tighten the sealing ring after it is fully immersed in the clarifying solution.With the outlet of the top piece is opened, slowly move the adapter down until all bubbles are drained.
  Note: This operation is only applicable to BXK 100 and above columns.Flushing the inner wall reduces the resin particles sticking between the seal ring and the column wall, avoiding the risk of leakage.

• When the bed height is 10cm, the flow rate can be set to 750cm/h. Open the bottom outlet, star tthe pump and run the setting flow rate until the bed is stability. Mark the bed height.

• Remove the packing reservoir (if any), lower the adapter to about 0.5cm above the resin surface, and continue to press the column using the above flow rate until the bed is completely consolidated, marking the consolidated bed height.

• Stop the pump, open the outlet of the top piece, close the outlet of the bottom piece, loosen the seal ring slightly, press the adapter to about 0.3cm below the marked position, tighten the seal ring, close the outlet, and complete the column packing.

3.2 Evaluation of Packing

• Test the column efficiency to check the quality of packing.The tests are required after the column packing, during the column working life and when the separation and purification performance is deteriorated. The method of the expressing the efficiency of a packed column is in terms of the height equivalent to a theoretical plate(HETP) and the asymmetry factor(As).
• Acetone or NaCl can be used as sample for the testing. Sample solution and eluent buffer can be prepared according to the following table.

| Acetone method| NaCl method
---|---|---
Sample| 1.0% (v/v)acetone in water| 0.8M NaCl in water
Loading| 1.0%CV| 1.0%CV
Buffer| Water| 0.4M NaCl in water
Flow rate| 30cm/h| 30cm/h
Monitor| UV280 nm| Conductivity

• Method for measuring HETP and As:
  According the UV curve or the conductivity curve to calculate the column efficiency(HETP), and the asymmetry (As):
  HETP=L/N
  N=5.54(VR/Wh)²
  Note: VR = retention volume
  Wh = half-peak width
  L = column height
  N = the number of theoretical plates(The units of VR and Wh should be  the same)
  As=b/a
  Note:****
  a= 1st half peak width at 10% of peak height
  b= 2nd half peak width at 10% of peak height
  

• Evaluation the column packing As a guideline, if  the value of HETP is less than 3 times the average particle size(d50)of the resin and the As is between 0.8~1.8, it is very efficient. The unsatisfactory results should be  analyzed and the column should be repacked .

3.3 Chromatographic method

• Sample

  • For complex protein mixed samples, the sample concentration should not be too low, the lower the binding capacity；However, for samples that specifically bind to the mediator ligand, there is no need to consider the sample concentration.
  • The sample concentration should not be too large. High concentration (greater than 30mg/mL) may cause fluctuations in pH and ionic strength, which affecting the binding. When the concentration is high, the sample can be diluted with binding buffer.
  • Pay attention to the viscosity of the sample. High viscosity samples will cause uneven flow rate during chromatography.
  • The sample solution needs to be centrifuged or filtered with a 0.45μm filter before loading, to avoid clogging the chromatography column or reducing the resolution efficiency and service life of the chromatography column.

• Binding buffer

  • The low pH binding buffer can promote protein binding. Generally, the pH range is between 5.5-8.5.
  • Low ionic strength balance buffer can promote protein binding, the concentration of binding buffer is preferably between 5-50mmol/L.
  • The presence of metal ions will also enhance protein binding capacity. Adding 0.1-10mmol/L of metal ions (such as Mg2+、 Ca2+、Zn2+、Mn2+、Cu2+、Co2+、Fe2+、and Al3+) to the buffer can enhance protein binding.

• Flow rate：The flow rate of 90 ~ 500cm/h is generally selected according to the height of the column. The larger the column height, the slower the flow rate.

• Sample preparation: In order to prevent blocking of the column, the sample needs to be filtered by microporous membrane of 0.45μm before loading, the pH and conductivity of the sample are adjusted to be consistent with the equilibration buffer. The loading volume was determined according to the impurity content in the sample and the binding capacity of Diamond Blue.

• Equilibrium: Clean the column with a balance buffer until the UV absorption is reduced to an appropriate value.

• Sample loading: sample loading shall be carried out according to the set procedure.

• Elution: One or several of the following methods to elute the bound protein can be used:

  •  Change the ionic strength of the elution buffer by increasing the concentration of salts (KCl and NaCl) in the buffer.
  • Increase the pH of the elution buffer.
  • Change the polarity of the elution buffer, such as adding 50% ethylene glycol, 10% dioxane, or other organic solvents.
  • Add appropriate concentrations of specific ligands such as enzyme substrates, enzyme substrate products, cofactors, inhibitors and activators.

• Regeneration: High pH (0.1M Tris, 0.5M NaCl, pH8.5) and low pH (0.1M NaAc, 0.5M NaCl, pH4.5) can be alternately cleaned to remove the strongly bound  roteins and make the chromatography column regenerate.

Cleaning-in-place(CIP)

With the increasing use of chromatography resin, the accumulation of contaminants on the chromatography column is also increasing. Cleaning-in- place can prevent the accumulation of contaminants and maintain a stable working state. Determine the frequency of CIP according to the degree of contamination of the resin (if the contamination is serious, CIP should be carried out after each use to ensure repeatability of the results).

• * Denatured protein：Wash 3~4CV with 0.5M NaOH, then wash 3-4CV with 70% ethanol or 2M potassium thiocyanate; or use 6M GuHCl to clean 2~3CV, immediately rinse with at least 5CV of equilibration buffer.

  • Strongly hydrophobic substances or lipids：Wash the column with 2-4CV of 70% ethanol or 30% isopropanol, and immediately rinse with at least 5CV of equilibration buffer.

Sterilization

Since the 20% ethanol with 0.1M KH2PO4 preservation solution does not does not have sterilization and depyrogenation,it is recommended that Diamond Blue can be treated with 70% ethanol for 12 hours to reduce the risk of microbial contamination before and during use. It can also be autoclaved at 121℃ for 15min.

Storage

Diamond Blue is supplied in 20% ethanol with 0.1M KH2PO4(pH 8.0)or 2% benzyl alcohol with 0.1M KH2PO4(pH 8.0). It should be stored in 20% ethanol with 0.1M KH2PO4(pH 8.0) and sealed at 2-8℃ after use, in order to prevent ethanol volatilization and microbial growth, it is recommended to replace 20% ethanol with 0.1M KH2PO4(pH 8.0)every 3 months.

Disposal and Recycling

Since Diamond Blue is difficult to degrade in nature, incineration is recommended to protect the environment.

Order information

SMSAA040 V2.01
021-58953301
www.bestchrom.com

References

• 博格隆（上海）生物技术有限公司

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