GSH Magnetic Beads for GST Tag Protein Purification letter
Contents
1. arator for separation then pipette the supernatant to a new centrifuge tube Take the tube off the magnetic separator and proceed to washing steps 4 5 Washing of Beads 1 Add 5 10 mL Buffer A to the centrifuge tube containing the beads shake them for 2 min perform a magnetic separation and pipette the elute to a new tube keep elution for a following test 2 Add 5 10 mL Buffer A to the centrifuge tube containing the beads to suspend them pipette the beads suspension to a anew tube to avoid nonspecific adherent proteins in the previous tube contaminating the target ones perform a magnetic separation and pipette the supernatant to the elute tube 4 6 Elution of Target Proteins 1 Add 2 5 mL Buffer B to the tube containing the beads users may change the elution volume to adjust the concentration of the target proteins if necessary close the tube cap tightly and mix and shake them at room temperature for 2 min perform a magnetic separation and collect the elution into a new tube This is the purified target protein samples 2 If necessary repeat the elution step 1 once again and collect the samples into a new tube to examine whether the target proteins have been eluted thoroughly Order amp Inquiry Tel 49 89 46148500 Fax 49 89 461485022 E mail eu order biotool com 4 7 Washing and Storage of Beads The beads can be used for purification again after washing treatment Additionally for a longtime storage user2. homogeneous suspension before use 4 Choose well qualified pipette tips and centrifuge tubes in case of the beads 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 manually 6 To analyze the purification process and optimize the protein purification procedure keep the removed supernatant elution assay for protein detection if necessary Order amp Inquiry Tel 713 732 2181 Fax 1 866 747 4781 _ E mail order biotool com 7 The product can be used multiple times as long as the same kinds of proteins are being assayed when purifying a different kind of protein sample choose the new beads to prevent the interactive contamination 8 The product should be used with the magnetic separator 9 The product can be stably stored at 4 C for two years 10 The product can only be used for research purposes 4 Reference Protocol for Gst tag Protein Purification The binding capability of the target proteins and the beads has a direct effect on the purification efficiency as does the preparation of the buffer on the recovery ratio and the purity of the target proteins Therefore users should design a preliminary experiment before a large scale purification to select the appropriate buffer which is recommended to include binding buffer Buffer A and elution buffer Buffer B A
3. nic ice bath to generate the crude protein samples Once the samples are homogenized add nucleases to the crude samples and place on ice for 30 min to degrade nucleic acids If the yield of the target proteins is expected to be low centrifuge the crude proteins to concentrate sample 2 Extracellular expressed proteins harvest the supernatant containing the extracellular expressed proteins and add equal amounts of Buffer A to generate the crude protein samples 3 Intracellular expressed proteins in animal cells wash animal cells once with PBS scrape into a tube and centrifuge Discard the supernatant and resuspend the cells with Buffer A containing 1 v v Triton X 100 or 1 v v NP 40 add a protease inhibitor lastly place the tube on ice for 10 min to generate the crude protein samples 4 3 Pretreatment of Beads The amount of beads used is determined by users calculations according to the target protein yield and the beads capacity For example for a protein expressed in E coli 1g wet weight of microbes can be obtained from 250 mL broth The yield of the target protein will come to 5 10 mg by the experimental estimation and users will need 10 mL 10 beads suspension for that target protein purification Details of this example are presented as follows 1 Homogenize the Biotool GSH Magnetic Beads fully with a vortex and pipette 10mL beads suspension to a centrifuge tube 2 Place the tube on the magnetic separat
4. of target proteins A2 You can try the following measures 1 Avoid ultrasonic lysis to keep the GST tag and the target protein intact 2 Add various protease inhibitors to prevent the protein degradation 3 Add 0 1 Tween 20 or 2 NP 40 to the samples and buffer to decrease the adsorption of the nonspecific proteins 4 Prolong the washing of the proteins and increase times of the washing 5 Elute the target proteins by GSH gradient concentration Order amp Inquiry Tel 49 89 46148500 Fax 49 89 461485022 E mail eu order biotool com
5. ol or 0 1 non ionic surfactant for three times place the tube on a vortex for 60 s each time perform a magnetic separation and discard the supernatant 2 Wash the beads with 10 mL 1xPBS for three times place the tube on a vortex for 60 s each time perform a magnetic separation and discard the supernatant After finishing the washing operations for Situation 1 and 2 if users need to use the beads again for protein purification wash the beads with Buffer A for 2 3 times as in the protein purification steps If storing them for a brief time wash the beads with 20 Ethanol for 2 3 times then add 20 v v Ethanol to the beads to a total volume of 10 mL and store them at 4 8 C Order amp Inquiry Tel 713 732 2181 Fax 1 866 747 4781 _ E mail order biotool com g biotool com Save time Save funding 5 Trouble Shooting Q1 How to increase the Recovery Ratio of Target Proteins A1 You can try the following measures 1 Prolong the incubation of the proteins and the beads 2 Add 1 10 mM DTT to the samples and buffer which helps increase the binding capability of certain GST fusion proteins and the beads Add various protease inhibitors to prevent the protein degradation Increase the amount of beads Prolong the elution of the proteins or increase times of the elution Use the just prepared fresh Buffer B to ensure efficient elution of the target proteins Oo BR Ww Q2 How to increase the purity
6. or discard the supernatant when the liquor becomes clear Note Make sure that every time you remove the supernatant by pipetting or pouring only when the tube is still attached to the magnetic separator 3 Add 5 10 mL Buffer A to the tube close the tube cap tightly oscillate the tube on a vortex for 15s to resuspend the beads place the tube on the separator for a magnetic separation and discard the supernatant 4 Repeat step 3 twice Attention in order to decrease the loss of beads during magnetic separation close the centrifuge tube cap tightly and keep the tube on the separator until the liquid becomes clarified Next invert the separator and the tube for several times by hand to wash the beads adherent to the cap by the clear liquid let the tube stand on the bench top until the liquid settles and 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 1g wet weight of microbes with 10 mL Buffer A and lysis This is the crude protein samples 2 Apply the samples to the tube containing the pretreated beads and close the tube cap tightly 3 Vortex the tube for 15 s then mix lightly and agitate it at room temperature for 20 30 min if necessary mix the tube at 4 8 C for 1 h to prevent the target protein degradation 4 Place the tube on the magnetic sep
7. s may choose different washing methods according to the usage situation of the beads Situation 1 When the beads are not repeatedly used for many times or the binding capability of the beads doesn t decrease obviously users may wash the beads by an alternative mode with high pH and low pH 1 Add 10 mL Buffer C 0 1 M Tris HCI 0 5 M NaCl pH 8 5 to the used beads place the tube on a vortex for 60 s perform a magnetic separation and discard the supernatant 2 Add 10 mL Buffer D 0 1 M Sodium Acetate 0 5 M NaCl pH 4 5 place the tube on a vortex for 60 s perform a magnetic separation and discard the supernatant 3 Repeat step 1 and 2 above twice Situation 2 When the beads are repeatedly used for many times the binding capability of the beads and the target proteins will decrease due to the precipitation denaturation or accumulation of the nonspecific adherent proteins Users may adopt the following methods to wash the precipitates or denatured proteins from the beads 1 1 Wash the beads with 5mL 6 M Guanidine Hydrochloride twice place the tube on a vortex for 60 s each time perform a magnetic separation and discard the supernatant 2 Wash the beads with 10 mL 1xPBS for three times place the tube on a vortex for 60s each time perform a magnetic separation and discard the supernatant In order to remove the hydrophobic conjugated substances wash the beads as follows 1 Wash the beads with 5 mL 70 Ethan
8. sample protocol depicting a GST fusion protein purification procedure with a relatively strong binding capability is provided below 4 1 Preparation of Buffer Buffer A Binding Buffer 140 mM NaCl 2 7 mM KCI 10 mM Na2HPO4 1 8 mM KH2P0O4 pH 7 4 Buffer B Elution Buffer 50 mM Tris HCl 10 mM GSH pH 8 0 Preparation Method Add 0 307g GSH to 100 mL 0 1M Tris HCl then adjust pH to 8 0 with 0 1 M HCI and add deionized water to a total volume of 100 mL Note 1 GSH is oxidized quickly so prepare Buffer B immediately before use 2 The binding capability between various GST fusion proteins and the beads is different Most GST fusion proteins can be eluted by Buffer B which contains 10 mM GSH A few GST fusion proteins which have a relatively strong binding capability may require prolonged elution increased time of the elution or increased concentration of GSH in Buffer B 3 To increase the stability of the target proteins users may add 1 5 mM EDTA 1 10 mM DTT 0 1 1 Triton X 100 or 0 1 1 Tween 20 to Buffer A and Buffer B 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 three samples as follows 1 Intracellular expressed proteins in bacteria yeasts and other cells dilute the cells with Buffer A and add a protease inhibitor like BiotoolTM Protease Inhibitor Cocktail lysis cells in an ultraso
9. v biotool com Save time Save funding GSH Magnetic Beads for GST 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 Cat Cat B23700 B23701 B23702 B23703 B23704 50 mL 5 mL X10 GSH Magnetic Beads for 500 ul 5 mL 10 mL 25m Gst Tag Protein 5 mL X2 5mL X5 Purification 1 Diameter of the bead is about 50 um 100 um 2 Amount of GSH Ligands is 20 30 umol mL 3 Binding capacity to proteins is about 5 10 mg mL pure beads 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 4C 30C 5 GSH Magnetic Beads for Gst Tag Protein Purification is stable in 70 Ethanol 6M Guanidine Hydrochloride 0 1M NaOH and 0 1M Acetic Acid at room temperature for 1h 2 Storage Information GSH Magnetic Beads for Gst 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 4C 30C and it is suggested to be stored at 4 Cfor 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 when using and storing the beads 3 Shake the beads thoroughly to keep them a
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