Phương pháp cô protein


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em chào anh chị
Em đang làm về biểu hiện, có lễ hàm lượng prrotein của em quá ít nên không thấy trên bản điện di, các anh chị có protocol vê cô protein thì share cho em với
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[FONT=Verdana, Arial, Helvetica, sans-serif]How can I concentrate protein in a dilute solution?[/FONT]

[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]As you near the end of your LSB607 protein purification scheme, there is a good chance that your final sample will comprise a dilute solution of relatively pure enzyme which you eluted from a chromatography column. [/SIZE][/FONT][FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]If you wish to analyse your enzyme on a ployacrylamide gel, you now may face the problem of not being able to load sufficient material on to the gel since, for a Bio-Rad Mini-Protean gel, the maximum volume you can load into a single lane will be 20 µL (plus 5 µL of sample buffer which must be added). The 20 µL volume of sample which you assay must contain between 5 and 20 µg protein (or even more if your sample is relatively impure), otherwise you may not see your protein in a Coomassie stained gel. If your protein concentration is below 300 µg/mL, you will need to concentrate your enzyme solution.[/SIZE][/FONT]
[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Your options are:[/SIZE][/FONT]

  • [FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]precipitate the protein and resuspend in a smaller volume (but then you must remove the salt by gel filtration or dialysis - annoying when you are dealing with very small volumes).[/SIZE][/FONT]
  • [FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]filter the solution through a microconcentrator and collect the protein in a much smaller volume (very quick and easy to do).[/SIZE][/FONT]
[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Regardless of the option you choose, you will need to repeat your protein assay after either of these procedures to ensure you load the correct amount onto a PAGE gel!!![/SIZE][/FONT]
[FONT=Verdana, Arial, Helvetica, sans-serif]Microconcentrators[/FONT]

[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Microconcentrators are ideal devices to use to concentrate small volumes and, at the same time, remove contaminating salt from protein-containing solutions. The filter insert is placed in an outer tube which collects the filtrate for convenient storage. The proteins collected from the filter insert by inverting the indert and collecting the proteins by a second spin.[/SIZE][/FONT]
[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Microconcentrators are very quick and easy to use, especially as a final concentration step in protein purification. In addition to concentrating proteins, these small filter units are ideal for concentrating and purifying proteins, antibodies, nucleic acids (alternative to EtOH precipitation); desalting and buffer exchange; removal of primers, linkers and unincorporated label; protein removal; DNA extraction from LMP agarose; DNA and RNA extraction from polyacrylamide gel; PCR purification; and detergent removal from small volumes of material.[/SIZE][/FONT]
[FONT=Verdana, Arial, Helvetica, sans-serif]Amicon and Microcon™ centrifuge filter units from Millipore[/FONT]

[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Millipore Corp market a wide variety of filtration media, including the small amicon Microcon filter devices. These devices simply and efficiently concentrate and desalt macromolecular solutions of 10 - 500 µL volume using a microfuge which can accept 1.5 mL Eppendorf tubes. [/SIZE][/FONT]
[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Microcon™ [/SIZE][/FONT][FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]


[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Millipore's Amicon Ultra product line centrifuge filter devices enable a single 15 min spin recovery of filtrates from up to 4 mL sample using either swing-out rotors (best) or fixed angle rotors which can accomodate a 15 mL conical bottomed tube. In these larger devices, filtrates are resuspended by pipetting up and down in a small volume of buffer.[/SIZE][/FONT]
[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Ami[/SIZE][/FONT][FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]con™


[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Using a Millipore Microcon column[/SIZE][/FONT]
  1. [FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Insert a sample reservoir into a vial.[/SIZE][/FONT]
  2. [FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Pipette protein solution (max volume 0.5 mL) into the sample reservoir without touching the membrane at the base of the reservoir. Seal with the attached cap.[/SIZE][/FONT]
  3. [FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Place the assembly in microfuge with the cap strap hinge facing the centre of the rotor. Ensure there is a counter balance in the opposite rotor position![/SIZE][/FONT]
  4. [FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Spin for the appropriate time (see table above) at room temperature.[/SIZE][/FONT]
  5. [FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Remove the assembly from the centrifuge and place the sample reservoir UPSIDE DOWN in a second, clean vial.[/SIZE][/FONT]
  6. [FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Centrifuge for 3 minutes at 1,000 x g (or a BRIEF PULSE in a MICROFUGE) to transfer the protein concentrate to the vial. Note: top speed in a microfuge is 12,000 - 14,000 x g! [/SIZE][/FONT]
  7. [FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Remove from centrifuge and discard (or re-use) the sample reservoir .The same column can be used as many as three times, allowing you to filter up to 1.5 mL of a dilute solution with a recovery of >95% of the protein following the recovery spins![/SIZE][/FONT]
  8. [FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Seal the cap and store your protein.[/SIZE][/FONT] [FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]For further information on the Microcon filters, consult literature available from the Millipore website.[/SIZE][/FONT]
[FONT=Verdana, Arial, Helvetica, sans-serif]VectaSpin™ centrifuge filter units from Whatman (GE Healthcare)[/FONT]

[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Whatman market a range disposable centrifuge filter devices which can be used in microfuges and larger tubes which may be spun in a bench-top (clinical) centrifuge. [/SIZE][/FONT]

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[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]

[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-1]VectaSpin™ Micro VectaSpin™ 3[/SIZE][/FONT][FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-1] VectaSpin™ 2
[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]For further information on the VectaSpin filters, consult literature available from the Whatman (GE Healthcare) website.[/SIZE][/FONT]
[FONT=Verdana, Arial, Helvetica, sans-serif]Microsep™ centrifugal devices from PALL Life Sciences[/FONT]

[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]PALL Life Sciences market Nanosep™ (500 uL) and Microsep™ (3.5 mL) centrifugal devices for concentrating and/or desalting proteins, nucleic acids and viruses. 100X concentration of proteins with over 90% recovery is possible using any fixed angle rotor that accepts 17 x 100 mm tubes and is capable of 3,000 - 7,500 x g. The device has a deadstop to prevent samples from spinning to dryness. Microsep MF™ with 0.2um and 0.45um Bio-Inert Membrane devices also are available.[/SIZE][/FONT]
[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]

[FONT=Verdana, Arial, Helvetica, sans-serif][SIZE=-2]Contact PALL Australia for further details. [/SIZE][/FONT]
Hôm nọ mình dùng cái Centricon MWC 50k Da để thử trên mẫu của mình. Kết quả thật bất ngờ (nhưng thú vị) là trong flowthrough chỉ có protein of interest với MW 11kDa. Chả trách nó bị mốc meo trong lab mấy năm.
Với mẫu có Vol nhỏ sau khi cô, cái Desalting column anh Cherry Pham giới thiệu mình thấy dùng được.
Microconcentrators là thi t b lý t ng s d ng t p trung kh i l ng nh và, ng th i, lo i b ô nhi m mu i t protein có ch a các gi i pháp. Các chèn b l c c t trong m t ng bên ngoài mà thu th p các vâ t c lo c cho vi c l u tr thu n ti n. Các protein thu c t vi c chèn l c b i ngh ch o các indert và thu th p các protein c a m t spin th hai.
Bạn biểu hiện protein từ hệ thống tế bào nào? Nếu từ E.coli thì còn phụ thuộc nhiều yếu tố: phương pháp biểu hiện chưa tối ưu, gen biểu hiện không over expression, phương pháp phá vỡ tế bào thu dịch protein chưa hiệu quả...
Bạn có thể sử dụng phương pháp tủa muối Ammonium sulfate hoặc tủa bằng acetone (protocol trên mạng rất nhiều) hoặc mua [FONT=&quot]Màng lọc cô đặc tinh sạch Amicon Ultra-15 Centrifugal Filter Units[/FONT][FONT=&quot] [/FONT]của Millipore để cô đặc protein.
mọi người cho Em hỏi muốn tổng hợp Protein bằng các chất hóa học thì tổng hợp từ những chất gì a? Em mới vào mong Anh Chị giúp đỡ ạ.


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