Nickel Magnetic Beads For His Tag Protein Purification letter
Contents
1. another tube for further assaying 2 Repeat the step 1 once again 3 Add 10 mL Buffer B to the tube containing the beads to resuspend them pipette the suspension to another tube to avoid nonspecific adherent proteins perform a magnetic separation and pipette the supernatant to the elute tube 4 6 Elution of Target Proteins 1 Add 2 10 mL Buffer C Elution Buffer adjust the volume based on concentration of the target proteins if necessary invert gently several times to resuspend the beads perform a magnetic separation and collect the elution into a new tube This is the purified target protein samples 2 If necessary repeat the step 1 once again and collect the samples into another tube to examine whether the target proteins have been eluted thoroughly 4 7 After treatment of Beads 1 Add 5 mL Buffer C to the tube containing the beads invert several times to suspend the beads perform a magnetic separation and remove the supernatant 2 Repeat the step 1 twice 3 Add 5 mL deionized water to the tube invert several times to suspend the beads perform a magnetic separation and remove the supernatant 4 Repeat the step 3 above twice 5 Add 20 v v ethanol to the beads for a total volume of 5 mL store the beads at 4 30C 4 C for a long storage for the next purification of the same proteins Order amp Inquiry Tel 49 89 46148500 Fax 49 89 461485022 E mail eu order biotool com 4 8 Regenera2. here depicting a His tag protein purification procedure with a relatively strong binding capability Figure 1 4 1 Preparation of Buffer Buffer A Binding Buffer 20 mM Sodium Phosphate 500 mM NaCl 5 50 mM Imidazole pH 7 4 Buffer B Washing Buffer 20 mM Sodium Phosphate 500 mM NaCl 50 100 mM Imidazole pH 7 4 Buffer C Elution Buffer 20 mM Sodium Phosphate 500 mM NaCl 500 mM Imidazole pH 7 4 The Buffer system above is apropriate for most His tag protein purifications Nonspecific binding can be reduced and binding capacity and purity of the target proteins can be increased by adding Imidazole into Buffer A For the first trial add 5 10 mM Imidazole and adjust the concentration of Imidazole in buffer according to purification results Additionally users may add other ingredients to the buffer such as Glycerin and surfactants if necessary Order amp Inquiry Tel 49 89 46148500 Fax 49 89 461485022 E mail eu order biotool com 4 2 Preparation of Samples This User Manual provides preparation of samples as follows 1 Intracellular expressed proteins in bacteria yeasts and other cells dilute the cells with Buffer A supplemented with a protease inhibitor like BiotoolTM Protease Inhibitor Cocktail perform cell lysis in an ultrasonic ice bath to generate the crude protein samples Once the samples are thick add nuclease and place on ice for 30 min to degrade the nucleic acids If the yield of the target pro
3. ecovery ratio and purity of the target proteins users may optimize the purification procedures according to the following parameters The concentration of Imidazole in the samples and Buffer A and the ingredients and concentration of the other reagents The treatment modes volume and concentration of the samples The amount of beads used in purification The incubation temperature and time of the samples and the beads Time and the frequency of washing the beads The concentration of Imidazole time the volume and the frequency when eluting the target proteins How to increase the Recovery Ratio of Target Proteins Decrease the concentration of Imidazole in the samples and Buffer A Add surfactants to the samples and the buffer Add protease inhibitors to prevent the protein degradation Increase the amount of beads used Prolong the incubation of the proteins and the beads Prolong the elution of the proteins or increase times of the elution Order amp Inquiry Tel 49 89 46148500 Fax 49 89 461485022 E mail eu order biotool com Q3 How to increase the purity of target proteins A3 You can try the following measures 1 A aE Increase the concentration of Imidazole and NaCl in the samples and Buffer A Add surfactants to the samples and the buffer Add protease inhibitor to prevent the protein degradation Prolong the washing of the proteins or increase times of the washing Elute the target proteins b
4. g biotool com Save time Save funding Nickel Magnetic Beads for His Tag Protein Purification Notice Magnetic beads should be stored at 4 C do not freeze Do not dry the magnetic beads 1 Package Information Component Cat Cat Cat B23600 B23601 B23602 500 ul 5 mL 10 mL 5 mL X2 Cat Cat B23603 B23604 25m 50 mL SmL X5 _ 5 mL X10 1 Diameter of the bead is about 50 um 100 um 2 Metal ions ligand is Ni2 and density of metal ions is 30 50 yu mol mL beads 3 Binding capacity to proteins is about 3 4 mg mL 10 beads suspension The protein binding capacity is provided as a reference True binding capacity is correlated with the nature of the target proteins 4 Working temperature is between 4 C 30 C Nickel Magnetic Beads For His Tag Protein Purification 2 Storage Information Nickel Magnetic Beads For His Tag Protein Purification is provided as 10 v v beads suspension 1 mL suspension contains 100 uL beads in the storage buffer of 20 Ethanol This product can be stored at 4 C 30 C and it is suggested to be stored at 4 C for long term stability 3 Notices for This Product 1 Read the User Manual carefully before the first use 2 Avoid freezing drying and high speed centrifugation during use and storage of beads 3 Shake the beads thoroughly to keep them in a homogeneous suspension before use 4 Choose well qualified pipette tips and centrifuge tubes
5. in case of bead loss from adherence or leakage when mixing 5 When mixing the beads and the solutions aspirate the beads with the pipette repeatedly or vortex briefly if the solution is too thick to resuspend the beads manually 6 To analyze the purification process and optimize the protein purification procedure keep the removed supernatant elution separated by the magnetic beads and assay for detection if necessary Order amp Inquiry Tel 713 732 2181 Fax 1 866 747 4781 _ E mail order biotool com 7 The beads can be reused but it is necessary to regenerate the beads when the purification capability decreases 8 Purify the same kinds of proteins when reusing the beads choose new beads when purifying different proteins 9 This product should be used with the magnetic separator 10 This product can be stably stored at 4 C for two years 11 The product can only be used for research purposes 4 Reference Protocol for His tag Protein Purification The binding capability of the target proteins and the metal beads has a direct effect on the purification efficiency The preparation of the buffers has a similar effect on the recovery ratio and the purity of the target proteins Users should design a preliminary experiment before a large scale purification to select the appropriate buffer which should include binding buffer Buffer A washing buffer Buffer B and elution buffer Buffer C A reference is provided
6. ration again and remove the supernatant repeat the deionized washing step 3 5 times until the eluate becomes neutral Add 1mL Recharge Buffer and invert several times to resuspend the beads spin and mix them at room temperature for 20 min perform a magnetic separation and remove the supernatant Add 1 mL deionized water and invert several times to resuspend the beads perform a magnetic separation and remove the supernatant repeat this step four times Order amp Inquiry g Tel 713 732 2181 Fax 1 866 747 4781 E mail order biotool com 4 Washing amp Elution 7 biotool com Save time Save funding Resuapend Cell lysis l Treatment of Samples ae a i Add Buffer 3 Binding of Target Proteins and Beads p Pretreatment of Beads Turn over and Mix for 30 min after Resuspension m Magnetic seperation Adel buffer B Collect supernatant qe Resuspend and H Repeat for three tines omogenize Collect supernatant d Add buffer C Resuspend and Homogenize gt a J rae tht beads Target Protein F Collect o supernatant Figure1 Procedures of the Nickel Magnetic Beads For His Tag Protein Purificatio 9 Q1 A1 a a E 2 Q2 You can try the following measures Dop e amp oa Trouble Shooting How to optimize the protein purification procedure The operation protocols above are appropriate for purifying most His tag proteins To increase the r
7. tached to the magnetic separator 3 Add 5 mL Buffer A to the tube above manually turn the tube and resuspend the beads place the tube on the separator for magnetic separation and discard the supernatant wash the beads twice Note in order to decrease loss of beads during the magnetic separation close the centrifuge tube cap tightly and keep the tube on the separator until the liquid becomes clear Manually invert the separator and the tube several times to wash the beads adherent to the cap by the clarified liquid let the tube stand until the liquid becomes clear again Order amp Inquiry Tel 713 732 2181 Fax 1 866 747 4781 _ E mail order biotool com g biotool com Save time Save funding 4 4 Binding of Target Protein and Beads 1 Suspend 2g wet weight of microbes in 10 mL Buffer A for lysis this is the crude protein samples Add samples to the tube containing the pretreated beads vortex for 15s 2 Place the tube on a shaker agitator mix at room temperature for 20 30 min if necessary mix the tube at 4 C for 1 h to prevent the target protein degradation 3 Place the tube on the magnetic separator pipette the supernatant to a centrifuge tube for further assaying Wash the beads and sample 4 5 Washing of Beads 1 Add 10 mL Buffer B Washing Buffer to the centrifuge containing beads invert several times to resuspend the beads perform a magnetic separation pipette the washing supernatant to
8. teins is expected to be low centrifuge the crude proteins to concentrate them 2 Extracellular expressed proteins harvest the supernatant containing the extracellular expressed proteins and add equal amount of Buffer A to generate the crude protein samples 3 Intracellular expressed proteins in animal cells wash animal cells once with PBS centrifuge and discard the supernatant resuspend the cells with Buffer A containing 1 v v Triton X 100 or 1 v v NP 40 attached table 1 add a protease inhibitor lastly place the tube on ice for 10 min to generate the crude protein samples 4 3 Pretreatment of Beads Usually the amount of beads used is determined by users calculations according to the target protein yield and bead capacity For example for a protein expressed in E coli 2g wet weight of microbes can be obtained from 500 mL broth The yield of the target protein will be 10 20 mg by the preliminary experimental estimation so 5 mL beads suspension is recommended for the target protein purification Details of this example are presented as follows 1 Homogenize the Nickel Magnetic Beads by vortexing and pipette 5mL beads suspension to a centrifuge tube 2 Place the tube on the magnetic separator pipette and discard the storage buffer when the liquid becomes clean remove the tube from the separator Note Make sure that every time you remove the supernatant by pipetting or pouring only when the tube is still at
9. tion of Beads After three uses the binding capability of the beads to the target proteins may decrease it is recommended to regenerate the beads Buffers needed Stripping Buffer 20 mM Sodium Phosphate 500 mM NaCl 100 mM EDTA pH 7 4 Bead Washing Buffer optional 0 5 M NaOH 2 M NaCl Recharging Buffer 100 mM NiSO4 this chemical reagent may lead to an allergic reaction so pay close attention when using it Example Take 1 mL 10 v v beads suspension for regeneration are as follows 1 Treatment by alkali optional step Perform a magnetic separation of the beads suspension remove the supernatant remove the tube from the separator add 1 mL deionized water to the tube and invert several times to resuspend the beads perform the separation again and remove the supernatant Add 1 mL Stripping Buffer and invert several times to resuspend the beads spin and mix them at room temperature for 5 min perform a magnetic separation and remove the supernatant repeat this step once Add 1 mL deionized water and invert several times to resuspend the beads perform a magnetic separation and remove the supernatant repeat this step twice Add 1 mL Bead Washing Buffer and invert several times to resuspend the beads spin and mix them at room temperature for 5 min perform a magnetic separation and remove the supernatant add 1 mL deionized water and invert for several times to resuspend the beads perform the sepa
10. y Imidazole gradient concentration Attached table1 Solvent Tolerance Solvent Category Solvent Name Concentration Tolerance Before adding solvent DTE 5mM wash the beads with the non DTE 5 mM reducing Reductant B solution 20 mM mercaptoethanol first Avoid DCEP 5 mM treating the beads with the Reduced 10 mM reductant Glutathione for a long time Urea 8M Denaturant Guanidine 6M Hydrochloride Triton X 100 2 Tween 20 2 Surfactant NP 40 2 Cholate 2 CHAPS 1 Sodium 50 mM Phosphate pH 7 4 HEPES 100 mM Tris HCl pH 7 4 100 mM Buffer Tris Acetate pH 100 mM 7 4 MOPS pH 7 4 100 mM Sodium Acetate 100 mM pH 4 0 Imidazole 500 mM Ethanol 20 Other Buffers NaCl 1 5 M Na2SO4 100 mM Glycerin 50 Order amp Inquiry Tel 713 732 2181 Fax 1 866 747 4781 E mail order biotool com we biotool com Save time Save funding Order amp Inquiry Tel 49 89 46148500 Fax 49 89 461485022 E mail eu order biotool com
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