Sepharose-4-Fast-Flow-matrix

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1、Sepharose 4 Fast Flow matrixSepharose 4 Fast Flow is a highly cross-linked agarose matrix. In its preactivatedCNBr form, it offers much improved performance when comparedwith the well established CNBr-activated Sepharose 4B. The Sepharose 4Fast Flow matrix has higher rigidity and can thus be run at

2、high flow rates(see Table 1).The higher mechanical strength of the cross-linked matrix makes it wellsuitedfor use in large columns. Scaling up a purification developed on CNBractivatedSepharose 4 Fast Flow is therefore simple and more predictable.The coupled product is stable at low pH, which is oft

3、en required for elutionfrom some immunoadsorbents.For applications that require operation at high pH, note that the amide bondformed when using the companion product NHS-activated Sepharose 4Fast Flow is stable up to pH 13 for normal use.2. CouplingCNBr-activated Sepharose 4 Fast Flow is supplied fr

4、eeze-dried in thepresence of additives. These additives need to be washed away at lowpH (pH-23) before coupling the desired ligand. The use of low pH (pH23)preserves the activity of the reactive groups, which otherwise hydrolyse athigh pH.In order to retain maximum binding capacity of CNBr-activated

5、 Sepharose 4Fast Flow prior to coupling the ligand, use cold (04 C) solutions. Thetime interval between washing and coupling must be minimised; thereforepreparations of all required solutions prior to coupling is recommended.1. Prepare the coupling solution, i.e. dissolve the ligand to be coupled in

6、a suitable coupling buffer, e.g. 0.1 M NaHCO3 pH 8.3 containing 0.5 MNaCl. For good coupling efficiency avoid unnecessarily dilute solutions(Recommended ratio of volumes, coupling solution/medium is 0.5:1). Thecoupling pH depends on the ligand. Normally pH in the range 79 is used.2. CNBr-activated S

7、epharose 4 Fast Flow is supplied freeze-dried withsugar additives and is washed initially with 1015 medium volumes ofcold 1 mM HCl, see Fig 1. Use small wash portions (e.g. 1 medium volume)and let the mixture equilibrate a few minutes during each washing step.After washing, determine the exact mediu

8、m volume obtained using e.g.centrifugation or PD-10 column (the medium volume may vary betweenexperiments).3. Mix the washed medium and coupling solution. Adjust pH to the desiredvalue. To obtain good reproducibility it is wise to adjust total reactionvolume to a fixed value with coupling buffer.4.

9、Coupling is normally very fast. At room temperature the reaction isusually completed after 24 hours. If coupling is performed at 4 C, it canbe performed overnight. It may be practical to follow the reaction usingUV-absorbance measurements.5. Wash away exess ligand with at least 5 medium volumes of c

10、oupling buffer.6. After coupling, non-reacted groups on the medium should be blocked.Transfer the medium to 0.1 M Tris-HCl buffer pH 8.0 or 1 M ethanolaminepH 8.0. Let it stand for 2 hours.7. Wash the coupled medium using alternate low and high pH.Recommended buffers are 0.1M acetate buffer pH 3-4 c

11、ontaining 0.5 MNaCl and 0.1 M Tris-HCl buffer pH 89 containing 0.5 M NaCl. A suitableprocedure could be 3x1 medium volume Tris HCl buffer followed by31 volumes acetate buffer. Repeat this cycle 36 times.8. The coupled medium is now ready for use. To prevent microbial growth,store in 20% ethanol for

12、example.Important Procedural Notes If the protein/peptide to be coupled is known to have free (reduced) sulfhydryls, go directly to the Procedure forImmobilizing a Protein/Peptide Having Free Sulfhydryls. Synthesized peptides, if used immediately after reconstitution,usually can be assumed to have s

13、ulfhydryls in the appropriate reduced state for coupling. If uncertain that free sulfhydryls exist in the protein/peptide to be coupled, use the Procedure for Reduction ofProtein/Peptide to Ensure Availability of Free Sulhydryls. As an alternative to this reduction procedure, especially forsmall pep

14、tides, use Immobilized TCEP Disulfide Reducing Gel (Product No. 77712) according to the productinstructions. To determine if free sulfhydryls are available on the protein/peptide, use Ellmans Reagent (Product No. 22582)according to the product instructions.Procedure for Reduction of Protein/Peptide

15、to Ensure Availability of Free SulfhydrylsA. Additional Materials Required Sample Preparation Buffer: 0.1 M sodium phosphate, 5 mM EDTA-Na, pH 6.0. Less than 10 ml is needed. Reductant: 2-Mercaptoethylamine (2-MEA, MW=113.61), Product No. 20408, for reducing disulfides Desalting Column: D-Salt Polya

16、crylamide Plastic Desalting Column, Product No. 43426, for removing reductant frompeptide after reducing disulfides Coupling Buffer: 50 mM Tris, 5 mM EDTA-Na, pH 8.5. Prepare a volume of buffer equal to 20 times the volume ofSulfoLink Coupling Gel to be used. UV-spectrophotometer or other device or

17、method to identify solution fractions containing peptide or protein.B. Reduction of Protein/Peptide1. Dissolve or dilute 1-10 mg protein/peptide per ml of Sample Preparation Buffer.Note: Sample size should not exceed 20% of the volume of the Desalting Column to ensure efficient desalting in Step 5.2

18、. Add 6 mg of Reductant (2-MEA) per ml of protein solution (results in 50 mM 2-MEA).3. Incubate mixture at 37C for 1.5 hours. (Begin Step 5 during this incubation).4. Cool mixture to room temperature (RT).5. Equilibrate Desalting Column by washing with 3 column volumes of Coupling Buffer.Note: All b

19、uffers to be run through a column should be degassed to avoid introducing air bubbles.6. Remove excess Reductant from the reduced sample by applying the mixture from Step 4 to the equilibrated DesaltingColumn from Step 5 and allowing the solution it to flow into the column.7. Add Coupling Buffer (ab

20、out equal to the desalting column bed volume) to the column and collect 0.5-1.0 ml fractions asthey drain from the column.8. Combine fractions containing the protein/peptide and proceed to the immobilization procedure.Procedure for Immobilizing a Protein or Peptide Having Free SulfhydrylsA. Addition

21、al Materials Required Disposable Column: Choose a column size appropriate for the volume of SulfoLink Coupling Gel to be used.Disposable Column Trial Pack (Product No. 29925) contains accessories plus 2 each or three different sized columns,appropriate for 1-10 ml gel bed volumes. Coupling Buffer: 5

22、0 mM Tris, 5 mM EDTA-Na, pH 8.5. If not already prepared for the Reduction Procedure, prepare avolume of buffer equal to 20 times the volume of SulfoLink Coupling Gel to be used. L-CysteineHCl: Product No. 44889 Wash Solution: 1 M sodium chloride (NaCl) Storage Buffer: Phosphate buffered saline (PBS

23、) or other suitable buffer containing 0.05% sodium azide (NaN3)B. Coupling Protein/Peptide to Gel1. Equilibrate SulfoLink Coupling Gel to room temperature (RT) and add an appropriate volume of gel slurry to an emptyDisposable Column. For example, add 2 ml of gel slurry to obtain a gel bed of 1 ml.2.

24、 Equilibrate column with 4 column volumes of Coupling Buffer.Note: Throughout the entire procedure, do not allow the gel bed to run dry; instead add additional solution or replace thebottom cap on the column whenever the buffer drains down to the top of the gel bed.3. Replace the bottom cap and add

25、the sample prepared in the Reduction procedure to the column, or dissolve thesulfhydryl-containing peptide in Coupling Buffer and add it to the column. Use 1 ml of protein solution per ml ofSulfoLink Coupling Gel. Retain a small sample of the peptide or protein solution for later determination of co

26、uplingefficiency.4. Replace the top cap and mix column (by rocking or end-over-end mixing) at RT for 15 minutes.5. Incubate the column at RT for an additional 30 minutes without mixing.6. Sequentially remove top and bottom column caps and allow the solution to drain from the column into a clean tube

27、.7. Place the column over a new collection tube and wash column with 3 column volumes of Coupling Buffer.8. Determine the coupling efficiency by comparing the protein/peptide concentrations (e.g, by absorbance at 280 nm) of theunbound fraction (Step 6) to the starting sample (Step 3).C. Blocking Non

28、specific Binding Sites on Gel1. Replace the bottom cap on column.2. Prepare a solution of 50 mM L-CysteineHCl in Coupling Buffer.3. Apply 1 ml of 50 mM cysteine solution to the column for each ml of gel.4. Replace the top cap and mix for 15 minutes at RT, then incubate the reaction without mixing fo

29、r an additional 30minutes at RT.D. Washing the Column1. Sequentially remove the top and bottom caps and allow the buffer to drain from the column.2. Wash the column with at least 6 column volumes of Wash Solution (1 M NaCl).3. Wash the column with 2 column volumes of degassed Storage Buffer.4. Repla

30、ce the bottom cap and add an additional 2 ml of Storage Buffer.5. (Optional): If using Pierce Disposable Column, insert the top porous disc, sliding it to within 1 mm of the gel bed.Note: A porous disc placed just above the gel bed prevents the column from drying out by automatically stoppingcolumn

31、flow when solution has drained down to the top of the gel bed. The disc also prevents resuspension of thepacked gel bed when adding solution to the column.6. The ligand is now covalently coupled to the support through its sulfhydryl groups and can be used for affinitypurification. Replace the top ca

32、p and store the column upright at 4C, or remove the bottom cap and proceed with StepA.3 of the General Protocol for Affinity Purification.General Protocol for Affinity Purification of ProteinNote: This protocol assumes use of a gravity-flow column with a gel bed volume of 2 ml. For columns with othe

33、r gel bedvolumes, adjust all solution (e.g., sample, wash, elution) volumes proportionately.A. Column Preparation1. Equilibrate the protein/peptide-coupled column to room temperature.2. Remove the top cap first to avoid drawing air into the gel bed.3. Remove the bottom cap and allow excess storage s

34、olution to drain from column.4. Equilibrate column by washing it with 6 ml of the same buffer in which the sample is diluted; e.g., Phosphate BufferedSaline (PBS, see Related Pierce Products).Note: All buffers to be run through a column should be degassed to avoid introducing air bubbles.B. Sample P

35、urification1. Apply up to 1.5 ml of sample to column and allow it to completely enter gel bed.Note: For samples 1.5 ml, repeat steps B.1-5 with 1.5 ml aliquots until the entire sample has been used. Alternatively,the gel can be removed from the column and incubated batch-wise with the entire sample

36、volume, then repacked as acolumn for steps B6-10.2. Apply 0.2 ml of sample buffer and allow it to enter the gel bed. Replace the bottom cap.3. Apply 0.5 ml of sample buffer to the column. Replace the top cap.4. Allow the column to incubate at room temperature for 1 hour.5. Remove the top cap, then t

37、he bottom cap from the column.6. Wash the column with 12 ml of sample buffer.7. Elute the bound protein by applying 8 ml of ImmunoPure IgG Elution Buffer (Product No. 21004 or 21009) or glycinebuffer (100 mM, pH 2.5-3.0).8. Collect 1 ml (or 0.5 ml) fractions.Note: 1 ml fractions can be neutralized b

38、y adding 100 l of 1 M Tris, pH 7.5 (or 50 l of 1 M Tris, pH 9).9. Monitor elution by absorbance at 280 nm.10. Pool fractions of interest and exchange into an appropriate storage buffer by desalting or dialysis.C. Regenerating and Storing the Affinity ColumnNote: Regenerate the column as soon as poss

39、ible after chromatography to prevent damage to the immobilized moleculebecause of the low pH of the elution buffer.1. Wash column with 16 ml of PBS to remove any residual protein and reactivate the gel.2. Equilibrate column with 8 ml of an appropriate degassed buffer containing 0.05% sodium azide.3. Replace the bottom cap and add 2 ml of degassed storage buffer to the column and cap the top.4. Store upright at 4C.文档可能无法思考全面，请浏览后下载，另外祝您生活愉快，工作顺利，万事如意!5 / 5