Data Sheet - BioVision
Contents
1. carefully at least 5 times and transfer the solution to a clean 1 5 ml microcentrifuge tube e Do the pipetting on the inner side surface of the membrane Centrifuge the microcentrifuge tube for 1 min at maximum speed This step will remove gel residues Transfer the protein containing solution to a clean 1 5 ml microcentrifuge tube Protein precipitation protocol for analysis by MALDI MS Add 1 10 by volume of MS buffer of your choice to the protein containing solution and mix properly For example add 70 ul of MS buffer to a 700 ul sample Incubate for 15 min at room temperature Add 1 5 by volume of 50 Trichloroacetic acid TCA and mix properly For example add 154 ul of 50 TCA to a 770 ul sample Incubate for 1 hour at 4 C Centrifuge the sample at 4 C for 30 min at 20 000 g Carefully descent the supernatant without disturbing the pellet 155 S Milpitas Blvd Milpitas CA 95035 USA T 408 493 1800 F 408 493 1801 www biovision com tech biovision com FOR RESEARCH USE ONLY BioVision Add 500 ul of ice cold acetone Incubate at 20 C for 30 min and centrifuge the sample at 4 C for 30 min at 20 000 g To increase protein precipitation yield incubate the samples over night at 20 C Carefully descent the supernatant without disturbing the pellet Air dry the pellet For mass spectrometric analysis resuspend the pellet in appropriate solution compatible with MALDI MS protein characteristic is important for dete2. each 0 1 ml of sample Low molecular weight salts and buffers e g Tris HCl and KPO equilibrate within 3 hours Equilibration times for viscous samples will be longer gt IMPORTANT User must determine exact equilibration times for the dialysis Change the dialysis buffer as necessary Pipette out the sample carefully from the DiaEasy Dialyzer tube to a clean microcentrifuge tube gt If sample volume has increased during dialysis let your sample evaporate on the bench top a fan increasing airflow across the membrane will speed up the process check every 10 min or less to prevent full evaporation and dryness Milpitas Blvd Milpitas CA 95035 USA T 408 493 1800 F 408 493 1801 www biovision com tech biovision com FOR RESEARCH USE ONLY BioVision Figure 1 Insertion of the DiaEasy Dialyzer tubes in the Figure 2 Supporting tray containing three DiaEasy provided supporting tray The arrow on the cap is Dialyzer tubes in a horizontal electrophoresis tank The positioned face up arrow on the cap is positioned face up and the two membranes of the DiaEasy Dialyzer tubes are in perpendicular to the electric field Vill Sample concentration by evaporation with DiaEasy Dialyzer tubes DiaEasy Dialyzer tubes are ideally suited for sample IX concentration via evaporation because of their dual membranes and large surface area Dialysis and concentration in the same device reduces protein loss Unlike closed
3. the tube Don t fill the tube with several gel slices for larger gel slices use more than one tube Place the DiaEasy Dialyzer tube in the provided tray see Figure 1 The supporting tray can comprise 1 4 DiaEasy Dialyzer tubes e IMPORTANT The arrow on the cap is pointing face up The two membranes of the DiaEasy Dialyzer tube must be in perpendicular to the electric field to permit the electric current to pass through the tube Place the supporting tray containing the DiaEasy Dialyzer tubes in a horizontal electrophoresis tank containing running buffer see Figure 2 e IMPORTANT Immerse fully the DiaEasy Dialyzer tube with the tray in the buffer Pass electric current usually at 80 150 volt until the nucleic acid exits from the gel slice see Tables 1 and 2 Optional Follow the DNA or RNA eluted out of the gel with a hand held UV lamp or table e IMPORTANT The electro elution time need to be adjusted for each individual sample Reverse the polarity of the current for 120 seconds This step will release the nucleic acid from the membrane Open the DiaEasy Dialyzer tube gently pipetting the solution up and down carefully at least 5 times and transfer the solution to a clean 1 5 ml microcentrifuge tube Do the pipetting on the inner side of the membrane Centrifuge the microcentrifuge tube for 1 min at maximum speed This step will remove gel residues Transfer the nucleic acid containing solution to a clean 1 5 ml microcentrifu
4. the supernatant without disturbing the pellet Wash the pellet with cold 70 ethanol Centrifuge the sample at 4 C for 30 min at 20 000 g Centrifuge the tube in the same orientation as previously to recover the DNA or RNA in a compact pellet e Air dry the pellet for 5 20 min gt Do not over dry the pellet e g by using a vacuum evaporator as this will make the DNA especially if it is of high molecular weight difficult to redissolve e Redissolve the DNA or RNA in a suitable buffer gt Use a buffer with pH gt 8 0 for redissolving as DNA does not dissolve readily in acidic buffers XI Protein Extraction from Polyacrylamide Gel with DiaEasy Dialyzer tubes IMPORTANT Fixation of proteins before electro elution e g fixation with methanol acetic acid etc is not recommended Fixation greatly reduces extraction yield A sensitive protein staining solution may be used as it permanently stains the gel without undue fixing of the protein Procedure Fill the DiaEasy Dialyzer tube with 0 8 ml of dH2O Incubate for at least 5 min Empty the tube e IMPORTANT Check carefully that no dH2O is leaking from the tube Absorbance of water by the dry membrane causes the decrease in water level e After staining the gel excise the gel slice containing the protein with a clean sharp scalpel Minimize the size of the gel slice by removing extra gel Maximum gel slice size should be 1 cm x 0 5 cm e Transfer the gel slice to a
5. 25 100 DiaEasy Dialyzer 15 ml MWCO 1 kDa K1010 10 25 100 DiaEasy Dialyzer 10 ml MWCO 12 14 kDa K1008 10 25 100 FOR RESEARCH USE ONLY Not to be used on humans 155 S Milpitas Blvd Milpitas CA 95035 USA T 408 493 1800 F 408 493 1801 www biovision com tech biovision com
6. BioVision FOR RESEARCH USE ONLY DiaEasy Dialyzer 800 pl MWCO 3 5 kDa bovis Catalog K1018 10 25 100 Store at RT 15 25 C Shelf Life 12 months for longer storage store at 4 C at relative humidity of 235 Introduction Biovision s DiaEasy dialyzer 3 5 kDa MWCO can combines two modes of action 1 dialysis or buffer exchange at sample volumes between 50 and 800 ul and 2 electro elution of macromolecules from polyacrylamide or agarose gel They can also be used for several other purposes as shown below This device allows rapid and high performance at either mode and extracts the macromolecules without any contamination They are ideal for purification of very small quantities of proteins up to 0 5 ug for automated protein sequencing peptide mapping and amino acid analysis The DiaEasy tube s membrane is ultra clean sulfur and heavy metal free and EDTA treated which makes it suitable for molecular biology experiments These tubes are autoclaved and bacteria free The DiaEasy tubes allow rapid secure and simple loading and recovery They are very high performance dialysis tubes and are the most convenient user friendly dialysis system on the market The sample quality after dialysis can be checked by several assays commonly used for proteins and nucleic acids Applications Dialysis or buffer exchange of small volumes 50 800 ul Extraction of proteins RNA DNA or oligonucleotides gt 20 nt from polyacrylamide agaro
7. DiaEasy Dialyzer tube Fill the tube with protein running buffer 0 7 0 8 ml Close the tube gently e Avoid air bubbles in the tube Do not fill the tube with several gel slices for larger gel slices use more than one tube e Place the DiaEasy Dialyzer tube in the provided supporting tray e The supporting tray can hold 1 4 DiaEasy Dialyzer tubes e IMPORTANT The arrow on the cap should point face up The two membranes of the DiaEasy Dialyzer tube must be in perpendicular to the electric field to permit the electric current to pass through the tube Place the supporting tray containing the DiaEasy Dialyzer tubes in a horizontal electrophoresis tank containing protein running buffer IMPORTANT Immerse fully the DiaEasy Dialyzer tubes with the tray in the buffer e Pass electric current usually at 100 volt until the protein exits from the gel slice 155 S Milpitas Blvd Milpitas CA 95035 USA T 408 493 1800 F 408 493 1801 www biovision com tech biovision com FOR RESEARCH USE ONLY E BioVision e Electro elution time is to be adjusted for each individual sample It takes at least 85 min for BSA protein to be electro eluted from a 10 SDS PAGE slice see Table 3 Reverse the polarity of the electric current for 120 seconds This step will release the protein from the membrane Open the DiaEasy Dialyzer tubes gently pipetting the protein containing solution up and down carefully at least 5 times and tran
8. g the gel excise the gel slice containing the protein with a clean sharp scalpel Minimize the size of the gel slice by removing extra gel Maximum gel slice size should be 1 cm x 0 5 cm Transfer the gel slice to a DiaEasy Dialyzer Tubes Fill the tube 0 7 to 0 8 ml with protein running buffer containing 250 mM Tricine DH 8 5 0 025 SDS and 25 mM Tris Base Close the tube gently e Avoid air bubbles in the tube Do not fill the tube with several gel slices for large gel slices use more than one tube Place the DiaEasy Dialyzer Tubes in the provided supporting tray e The supporting tray can hold 1 4 DiaEasy Dialyzer Tubes Place the supporting tray containing the DiaEasy Dialyzer Tubes in a horizontal electrophoresis tank filled with protein running buffer 250 mM Tricine pH 8 5 0 025 SDS and 25 mM Tris Base IMPORTANT Immerse fully the DiaEasy Dialyzer Tubes with the tray in the buffer Pass electric current at 150 volt until the protein exits from the gel slice e The electro elution time is to be adjusted for each individual sample It takes at least 2 hours for BSA protein to be electro eluted from a 10 SDS PAGE gel slice For other proteins from BSA increase electro elution time presented in Table 3 by 30 Reverse the polarity of the electric current for 120 seconds This step will release the protein from the membrane Open the DiaEasy Dialyzer Tubes gently pipetting the protein containing solution up and down
9. ge tube e Note Concentrate the extracted nucleic acid by standard concentration methods Elution Time Tables In this method the elution time depends on the size of the nucleic acid fragment the concentration of the gel the size of the gel slice the ratio of the polyacrylamide bisacrylamide and the applied voltage e IMPORTANT The electro elution time at the elution step needs to be adjusted for each individual sample 155 S Milpitas Blvd Milpitas CA 95035 USA T 408 493 1800 F 408 493 1801 www biovision com tech biovision com BioVision FOR RESEARCH USE ONLY Table 1 Minimum time needed to extract various DNA Table 2 Minimum time needed to extract DNA or RNA fragments from native or denatured 4 fragments from 1 agarose gel at 80 110 volt polyacrylamide gel 29 1 polyacrylamide bisacrylamide at 100 150 volt X DNAor RNA precipitation procedure e Add 0 1 volumes of 3 M Potassium acetate pH 5 2 and 0 7 1 volumes of isopropanol to the solution For example add 70 ul of 3 M Potassium acetate pH 5 2 and 500 700 ul isopropanol to a 700 ul sample Mix gently by inverting the tube several times gt Note addition of carrier e g 20 ug tRNA or 20 ug glycogen to the solution will increase the efficiency of precipitation Incubate at 20 C for 10 min To increase DNA or RNA precipitation yield incubate the samples overnight at 20 C Centrifuge the sample at 4 C for 30 min at 20 000 g Carefully discard
10. r at 4 C Centrifuge the sample at 4 C for 30 min at 20 000 g and carefully decant the supernatant without disturbing the pellet Add 500 ul of ice cold acetone Incubate at 20 C for 30 min and centrifuge the sample at 4 C for 30 min at 20 000 g To increase protein precipitation yield incubate the samples overnight at 20 C Carefully decant the supernatant without disturbing the pellet Air dry the pellet Resuspend the pellet in a suitable buffer solution or 0 1 M NaOH use at least 20 ul to perform resuspension XIV Related Products DiaEasy Dialyzer 800 ul MWCO 1 kDa K1017 10 25 100 DiaEasy Dialyzer 800 ul MWCO 6 8 kDa K1019 10 25 100 DiaEasy Dialyzer 3 ml MWCO 3 5 kDa K1012 10 25 100 DiaEasy Dialyzer 3 ml MWCO 6 8 kDa K1013 10 25 100 DiaEasy Dialyzer 3 ml MWCO 12 14 kDa K1014 10 25 100 DiaEasy Dialyzer 3 ml MWCO 25 kDa K1015 10 25 100 DiaEasy Dialyzer 20 ml MWCO 3 5 kDa K1000 10 25 100 DiaEasy Dialyzer 15 ml MWCO 3 5 kDa K1001 10 25 100 DiaEasy Dialyzer 20 ml MWCO 6 8 kDa K1003 10 25 100 DiaEasy Dialyzer 15 ml MWCO 6 8 kDa K1004 10 25 100 DiaEasy Dialyzer 10 ml MWCO 3 5 kDa K1002 10 25 100 DiaEasy Dialyzer 20 ml MWCO 12 14 kDa K1006 10 25 100 DiaEasy Dialyzer 15 ml MWCO 12 14 kDa K1007 10 25 100 DiaEasy Dialyzer 10 ml MWCO 6 8 kDa K1005 10 25 100 DiaEasy Dialyzer 20 ml MWCO 1 kDa K1009 10
11. rmination the appropriate solution followed by essential dilution step according to the protocols compatible with MALDI MS Use at least 20 ul to perform resuspension XIII Protein precipitation protocols used after isolation from the DiaEasy Dialyzer Tubes Trichloroacetic acid TCA precipitation procedure for protein Add equal volume of 20 TCA to the microcentrifuge tube containing the extracted protein solution and mix properly For example add 700 ul of 20 TCA to a 700 ul sample Incubate 60 min in 4 C Centrifuge at 4 C for 30 min at 20 000 g and carefully discard the supernatant Add 500 ul cold acetone and incubate at 20 C for 60 min Centrifuge the sample at 4 C for 30 min at 20 000 g To increase protein precipitation yield incubate the samples overnight at 20 C Discard supernatant and air dry the pellet Resuspend the pellet in 0 1 M NaOH or dH2O use at least 20 ul to perform resuspension If dH2O is used for resuspension incubate the sample for 5 min in 60 C resuspend the sample and incubate 5 min more at 60 C MS precipitation procedure recommended when protein bound SDS need to be removed Add 1 10 by volume of your choice of MS buffer to the protein containing solution and mix properly For example add 70 ul of MS buffer to a 700 ul sample Incubate for 15 min at room temperature Add 1 2 by volume of 20 TCA and mix properly For example add 385 ul of 20 TCA to a 770 ul sample Incubate for 1 hou
12. se or any gel matrix in any running buffer Extraction of protein protein DNA protein or RNA protein complexes Preparation of protein samples for MALDI MS Samples concentration High through put dialysis using a single beaker with several DiaEasy tubes on one floating pad Yield Recovery DNA or RNA from Agarose gel DNA or RNA from polyacrylamide gel Protein from SDS PAGE gel Kit Contents Components K1018 10 1018 25 K1018 100 Part Number Vi Vil 155 S User Supplied Reagents and Equipment Beakers and Buffers Storage and Handling Store all components of the kit at room temperature Read the entire protocol before performing the experiment Dialysis by DiaEasy Dialyzer Tubes Procedure Fill the DiaEasy Dialyzer Tube with 800 ul of dH2O0 Incubate for at least 5 min Empty the tube IMPORTANT Carefully check that no dH2O leaking from the tube Water absorption by the dry membrane causes decrease of water level Load sample into the DiaEasy Dialyzer Tube Close the tube gt IMPORTANT Sample volume should be in the range of 50 800 ul If small volume is used load the sample close to the inner membrane Place the loaded DiaEasy Dialyzer Tube in the supplied floating rack in a stirred beaker containing large volume usually 100 to 1000 fold that of the sample of the desired buffer The floating rack can hold 1 4 DiaEasy Dialyzer Tubes Adjust the stir bar speed Allow at least 30 min for
13. sfer the solution to a clean 1 5 ml microcentrifuge tube Do the pipetting on the inner side of the membrane Centrifuge the microcentrifuge tube for 1 min at maximum speed This step will remove gel residues Transfer the protein containing solution to a clean 1 5 ml microcentrifuge tube Important Notes Use the extracted protein directly Concentrate the extracted protein by standard concentration methods Precipitate the extracted protein by standard precipitation protocols Dialyze directly the extracted protein with a clean DiaEasy Dialyzer tubes Elution Time Table The elution time depends on the size of the protein molecule to be eluted the applied voltage the size of gel slice the ratio of the polyacrylamide bisacrylamide and the percentage of the polyacrylamide gel Electro elution time at the elution step was to be adjusted for each individual sample Table 3 Minimum time needed to extract different sized 19 26 45 55 66 85 95 proteins from 10 SDS polyacrylamide gel 29 1 a 5565 105 115 polyacrylamide bisacrylamide at 100 V 29 65 75 128 140 150 XII Protein Extraction from polyacrylamide Gel compatible with MALDI MS by DiaEasy Dialyzer Tubes Procedure Fill the DiaEasy Dialyzer tube with 0 8 ml of dH20 Incubate for at least 5 min Empty the tube IMPORTANT Check carefully that no dH20 is leaking from the tube Absorbance of water by the dry membrane causes decrease in the water level After stainin
14. system centrifuge type devices sample concentration can be easily monitored in the DiaEasy Dialysis tubes Procedure Place sample in the DiaEasy Dialyzer tubes or use already dialyzed sample and place it on the microtube rack stand Let samples evaporate on the bench top using fan to increase airflow across the membrane speed up evaporation process check every 10 min or less to prevent full evaporation and dry sample condition Concentrating by evaporation of water with sample small molecules buffer salts reducing agents etc will also get concentrated because no diffusion occurs gt IMPORTANT When evaporating water from your sample small molecules buffer salts reducing agents etc will also be concentrated DNA RNA extraction from gel with DiaEasy Dialyzer tubes This procedure is designed to extract DNA or RNA from polyacrylamide or agarose gels Procedure Fill the DiaEasy Dialyzer tube with 800 ul of dH20 Incubate for at least 5 min Empty the tube IMPORTANT Check carefully that no dH20 is leaking from the tube Absorbent of water by the dry membrane cause to the decrease in water level Excise the slice of gel containing the desirable DNA or RNA fragment with a clean sharp scalpel Minimize the size of the gel slice by removing extra gel Maximum gel slice size 1 cm x 0 5 cm Transfer the gel slice to a DiaEasy Dialyzer tube Fill the tube with 0 7 0 8 ml dH2O Close the tube gently Avoid air bubbles in
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