Operating Instructions
Contents
1. CH3 3NBH3 SBH CAS 16940 66 2 Sodiumborohydride NaBH Caution Schiff s base is reduced selectively Reduction force is weak and there is almost no damage given to protein Blocking operation is unnecessary Toxicity is low Solubility is low 1 3 of solubility to water Since reduction force is strong a formyl group is reduced In special protein use is impossible A S S binding is reduced The pre incubation of 1 to 2 hour is required The pre incubation of 1 to 2 hour is required Solution of SCBH should be made in a well ventilated area since a small amount of toxic gas will be generated upon solubilization m Ligand solution typically 1 20 mg ml in coupling buffer Coupling efficiency decreases in 20mg ml or more Note total mass coupled will be directly related to concentration see Fig 1 60 LI 100 a il _O Q gt E u 80 3 40 Ao O p gt D E ae O S p a LES e 60 30 Pe 3 VF og 4i 2 Cc a a sees gy 4 S jos o H oe F Taa a 40 3 9 20 ied f g 9 a f o 4 20 0 O 0 0 0 20 40 60 80 100 ligand nyg m Fig l The effect of the concentration of ligand in the coupling reaction O hy Glo Coupling O hy Glo Coupling ratio HAS Coupling HAS Coupling ratio www amsbio com amsbio info amsbio com General Coupling Procedure Table 1 The summary of operation Re2. MCellufine Operating Instructions Affinity Chromatography Media for ligand coupling Cellufine Formy Description Cellufine Formyl is an aldehyde activated support for the covalent immobilization of amine containing proteins and ligands As with all Cellufine products the base support consists of spherical cellulose beads which exhibit Superior rigidity and chemical stability relative to classical agarose gels Such mechanical strength allows for improved throughput at both the bench scale and on the production floor The exclusion properties of Cellufine Formyl are similar to those of 4 agarose gels Furthermore due to the robust internal structure and chemical linkage immobilized ligands show no appreciable leakage With the use of a condensation agent ligands proteins etc can be easily coupled via the reactive aldehyde moiety Physical Chemical Characteristics Base media porous cellulose spherical beads Particle Size ca 125 210 um Activated group Formyl aldehyde Formyl group conc 10umol ml gel 0 2M Acetate buffer oH3 0 containing 0 01 2 2 dithio bis pyridine 1 oxide Delivery condition Coupling reductive amination The coupling of protein ligands having primary amino groups to Cellufine Formyl proceeds via a Schiff s base intermediate followed by reduction with reducing agent NaCNBH SCBH CH3 3NBH3 JT MAB and NaBH SBH as illustrated below Preincubation H O Reducing agent O Qo
3. Rk a h Cu HNRS QK GR amp O A Q RH TT RN MAR Gy aR HH H H H O H Cellufine Formyl Ligand Schiff base Ligand immobilized Cellufine AMSBIO www amsbio com info amsbio com E UK amp Rest of the World HEE North America Germany oll Switzerland BSS 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 41 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 49 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 Materials Coupling buffer The buffer solution which does not contain a primary amine the range of pH 4 to 11 and concentration are about 0 2M a phosphate buffere an acetate buffere HEPES buffer solution etc can use m Blocking buffer 0 2M to 1M of Primary amine compounds in coupling buffer such as ethanol amine glycine and glycine ethyl ester etc or 0 2M to 1M of Tirs HCI buffer pH 7 to 8 is used m Reducing agent the most common reducing agents are sodium cyanoborohydride SCBH Trimethylamjneborane TMAB and sodium Borohydride SBH Ooo Advantage Eo pH SCBH Protein and aldehyde Toxic substance used at a draft chamber gt pH4 CAS 25895 60 7 compound is not Waste processing Sodjumcyanoborohydride reduced but a necessity NaCNBH3 TMAB CAS 75 22 9 Trimethylamjneborane
4. amsbio com Regeneration The regeneration protocol will depend on ligand stability In some cases a few bed volume washes with elution buffer containing 0 1 Tween 20 or Triton X 100 will be sufficient A similar wash with 6 M urea can also be effective Storage For storage of opened containers it is recommended that they be kept in a cold room 2 8 C Do not freeze Shelf Life 5 years from date of manufacture Product Ordering Information Catalogue No Pack Size Media type Cellufine Formyl 676 944 324 19853 19854 19855 676 944 335 AMSBIO www amsbio com info amsbio com e UK amp Rest of the World HEE North America m Germany Switzerland AS 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 41 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 49 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218
5. he inlet open to release air insert and affix the top adjuster assembly at slurry interface 5 Open the column outlet and begin pumping adsorption buffer at rate at least 20 higher than the operational flow rate 6 After the bed stabilizes close the column outlet Then with the inlet open reposition the end cell on top of the bed Operating Guidelines General Operation 1 Wash column with 5 bed volumes of elution buffer Equilibrate with 5 bed volumes of adsorption buffer Load sample Wash with 5 bed volumes of adsorption buffer a Elute sample with 5 bed volumes of elution buffer Recommended Buffers These conditions will depend on the ligand used However the following are generally useful for immuno affinity chromatography Adsorption buffer 20 mM phosphate 0 1 M NaCl pH 7 2 Depending on application other buffer ions may be used Elution buffer 0 1 M glycine pH 3 5 Sample Preparation and Load Prepare a 1 10 mg ml solution of sample in adsorption buffer Remove insoluble material by centrifugation or microfiltration Flow Rate The recommended linear velocity range for Cellufine Formyl is 20 150 cm h Chemical and Physical Stability The stability of the coupled gel will be limited by the ligand However the base gel is stable to most salts detergents chaotropic agents 0 1 N NaOH 0 1 N HCI and can be autoclaved at 121 C for 30 minutes at pH 7 0 www amsbio com amsbio info
6. n Fig 2 The effect of pH of coupling buffer on the amount of O hy Globulin 30 A hy Globulin 4 C ligand coupled to the gel HAS 30T eae HAS 4C pH of a coupling buffer Optimal pH changes with protein types OH stability and reaction efficiency of a ligand are taken into consideration Generally pH higher than pl goes up reaction efficiency See Fig 2 Schiff s base Formation and Reduction A Schiffs base is readily formed between the amine ligand and aldehyde gel Sodium cyanoborohydride specifically reduces this linkage to a C N bond minimally affecting the ligand Given such specificity sodium cyanoborohydride can be added at time zero In contrast if BaBH is used there should be a pre incubation period in order to allow Schiffs base formation Otherwise the aldehyde moiety will be prematurely reduced Reaction Temperature Lower temperature result in lower reaction rate and in some cases less efficient coupling As such recommended reaction time should be increase to 16 hours if refrigeration at 4 C is required Column Packing 1 Calculate volume required for the desired bed dimension keeping in mind that bed compression will occur during column packing Prepare a 40 60 v v slurry with the appropriate adsorption buffer With outlet closed pour the slurry into column Depending on the volume a filler tube may www amsbio com amsbio info amsbio com be necessary 4 With t
7. quired quantity per 1mL gelf Cellufine Formyl preservation solution is washing removed 1ml 0 7g to 0 8g wet g Coupling buffer Coupling buffer solution containing ligand 1 to 2 ml Refer to the Coupling Considerations for ligand concentration 3 Preincubation agitates gently for 1 to 2 hours jpreincubation is unnecessary when SCBH is used S ose Tomam ar nireongagers rn S O 100mg ml of 1ml ig S Coupling reaction agitates gently for 2 8 hours It depends for the temperature suitable for a reaction on the stability of a ligand Washing A reaction solution is removed by filtration or decantation washes in several times by coupling buffer 20ml The amount of coupling can be calculated by measuring the ligand concentration contained in reaction solution and washing solution Blocking reaction 1 2ml blocking solution is added to the washed gel and the same quantity as a reducing agent 4 is applied agitates gently for about 2 hours Washing A reaction solution is removed by filtration or decantation Washes in several times by 20ml of coupling buffer 2 or water Perform the following in a suitable mixing vessel 1 Estimate the required volume of medium As for the bottle of a product about 50 of Cellfuine slurry is contained 2 Preservation solution is removed by filtration or decantation Washes in several times by coupling buffer 10 to 20ml until an acetic acid smell is lost 3 Add the ligand sol
8. ution at a 1 to 2 times of gel volume This will form a total slurry volume approximately 2 to 3 times the original volume of medium If using SCBH preincubation is not necessarily required If using SBH blocking reaction is unnecessary Because SBH is for reducing aldehyde Formyl and turning it a hydroxyl group Note SCBH waste solution dispose of in a manner consistent with federal state and local regulations And please refer to MSDS of a SCBH manufacture company Coupling Considerations Ligand loading and biochemical activity are influenced by solute concentration pH reaction time and temperature The coupling conditions above will be appropriate for most applications Ligand Concentration Ligand loading is directly related to the ligand concentration See Fig 1 Using a 50 slurry 1 ml gel per 1 ml ligand solution and about 10 mg ml ligand concentration 60 coupling can www amsbio com amsbio info amsbio com be achieved in approximately 4 hours at room temperature Higher efficiency can be obtained with longer reaction times See Fig 3 100 pe o ae heer ete A z m x vec ee O 80 oO wv m J a I Sy me ee Ce A T 60 e e Cc 3 40 oes O hy Globulin 5 O HAS ee 0 5 10 15 20 reaction tire H 0 2 3 4 5 6 7 8 9 10 11 12 Fig3 The relation between the time for coupling reaction oH and the quantity of coupled protei
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