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DNA纯化手册,免疫性沉淀反应

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核心提示:General notes: 1) Endotoxin - you must assume that all standard laboratory glassware will be coated with loads of end

核心提示:OutlineImmunoprecipitation is a technique that permits the purification of specific proteins for which a antibody has

DNA纯化手册,免疫性沉淀反应。核心提示:IntroductionThis cheap and simplified protocol, based on Hansen et al., 1995, gives high yields of plasmid DNA as w

General notes:

Outline

Immunoprecipitation is a technique that permits the purification of specific proteins for which a antibody has been raised. This primary antibody is either already bound to agarose or can be bound to the protein A/agarose beads during the procedure in order to physically separate the antibody-antigen complex from the remaining sample.

Introduction

This cheap and simplified protocol, based on Hansen et al., 1995, gives high yields of plasmid DNA as well as high purity and is suitable for cloning, PCR, sequencing, site-directed mutagenesis and in vitro transcription, etc. It is an extremely good method for routine application and provides a good alternative when yields tend to be low due to low plasmid copy number for any number of reasons . Yields are at least 3 - 5 times higher than those obtained with commercial plasmid purification kits and often even ten-fold higher. Plasmid DNA can be recovered from drained liquids usually discarded when employing commercial kits. 30 - 50% more plasmid can be recovered if these are passed through a diatomaceous earth system.

The yield of plasmid using the protocol described here is higher than that obtained with commercial kits even if only using low or normal quality diatomaceous earth. From a 3 ml of E. coli culture overnight, 30-60 'micro'g of plasmid can be obtained at a purity of 1.8 to 2.0 . For a 50 ml overnight culture, 500 - 800 'micro'g of plasmid can be typically obtained.

The method has been successfully employed using plasmids ranging from about 3.0 to > 100 kb. The method basically employs two steps - alkaline Iysis of cells and elution of DNA from a home-made diatomaceous earth binding matrix . We used a Promega WizardTM minicolumn , but other brands/types of columns can also be used. Centrifugation in the protocol is carried out at 13,000 rpm on a minifuge unless otherwise specified.

Procedure

  1. 皇家赌场号hj85,Grow a 3 ml culture of E. coli overnight containing an appropriate antibiotic

  2. Harvest the cells by centrifugation for 2 min and suspend in 300 - 500 'micro'l of Suspension Solution at room temperature.

  3. Add 300 - 500 'micro'l of Lysis Solution, mix very gently and keep at room temperature for about 5 min

  4. Then add 300 - 500 'micro'l of Neutralising Solution. Invert gently several times and centrifuge for at least 7 min. Fresh diatomaceous columns should be prepared during this time . Although diatomaceous solutions store well, the columns don't.

  5. Carefully transfer the supernatant and mix with approximately the same volume of Binding Bufferin a syringe and apply the mixture to the top of a freshly made diatomaceous earth column . Once all the solution has been transferred, and no sooner, apply gentle suction in the same manner as described for preparation of the column.

  6. Add 1 ml of Washing Solution and gently drain in the same way. Then, place the column into an eppendorf tube a) and centrifuge for at least 3 min to make sure that all the Washing Solution is completely removed from the column. It is necessary to repeat step 6 twice .

  7. Place the washed and drained column into a new eppendorf tube and add 50 'micro'l of preheated MQ water or TE buffer to elute the DNA and place at room temperature for 10 min .

  8. Centrifuge the column for 1 - 2 min. Repeating step 7 - 8 two or three times elutes virtually all of the DNA.

a) For economic purposes, save the emptied eppendorf tube from step 4 for step 6.

Solutions required:

All solutions should be prepared in high quality deionised water suitable for molecular biology.

1. Suspension Solution

50 mM Tris-HCl, pH 7.5 - 8.0, containing 10 mM EDTA and 100 'micro'g/ml DNase-free RNase A.

Store at 4oC.

However, in the case of plasmid isolated from bacteria such as Xanthomonas spp. or Pseudomonas spp., producing exo-polysaccharide during culture, use either Suspension Solution containing 3 % NaCl or just 3 % NaCl. Alternatively, approximately 3 % NaCl could be added directly to a bacterial culture. Mix thoroughly before proceeding with step 2.

2. Lysis Solution

0.2 M NaOH containing 1 % SDS

3. Neutralising Solution

4 M potassium-acetate, pH 4.8

Place 23.55 g potassium acetate in measuring cylinder and fill to 66 ml mark with MQ water . Add 28.5 ml glacial acetic acid, mix and titrate with about 1.5 ml of concentrated HCl to pH 4.8. Top up to 100 ml with MQ water .

4. Binding Buffer

6 M guanidine hydrochloride

It is not necessary for guanidine hydrochloride to be dissolved in TE buffer as described in Hansen et al. as MQ water is equally good. 5 M or 4 M works well but 6 M is preferable. Anything less than 3 M gives poor results.

5. Washing Solution

80% isopropanol . Ethanol is generally good as a washing solution, except that isopropanol is cheaper.

6. TE Buffer

10 mM Tris-HCl, pH 8.5, containing 1 mM EDTA

7. Diatomaceous Earth Solution

The preparation of this solution is crucial. Suspend the diatomaceous earth at 50 mg/ml in water and leave to sediment for more than 3 hrs. Carefully discard as much of the water containing the white gelatinous colloidal suspension as possible, but leave the sediment intact. Repeat at least 3 times . If fine gelatinous matter is found during use, then discard the supernatant carefully and replace it with the same amount of water to maintain the same concentration of diatomaceous earth as above. Again, any milky suspension of diatomaceous earth should be removed as above. Even normal or low quality diatomaceous earth gives much better yields than any of the commercial kits tried. High quality diatomaceous earth is only necessary when an ultrapure plasmid preparation is required. We have not tested the difference between a highly pure plasrnid and an ultrapure plasmid preparation, but we think that the results will be the same as long as the plasmid purity is between 1.8 to 2.0

Preparation of the diatomaceous earth column

The diatomaceous earth solution should be resuspended thoroughly before use.

  1. Place a 2 - 5 ml syringe to a minicolumn and attach to a vacuum fitting, but not apply vacuum as yet!

  2. Load about 500 - 600 'micro'l of diatomaceous earth solution onto the column and apply suction. Once all the solution has been applied, watch the column from above and begin to apply gentle suction. Disconnect the vacuum immediately when the liquid phase disappears and the surface becomes solid. The column should look greyish white, with a thin brilliant white band at the bottom. If the column is brilliant white all the way up, the vacuum has been applied for too long. Dried columns don't bind DNA.

If you do not have a vacuum device or suitable setup, connect a syringe to the top of the column via the luer lock and apply pressure gently to obtain the same effect. Be sure to disconnect the syringe from the column before pulling back on the plunger. The column is now ready to be used in step 5 of the procedure section. The syringe can be reused after cleaning with MQ water or distilled water. The column can also be reused after appropriate cleaning as described below.

1) Remove the diatomaceous earth completely from the column.

2) Soak the column in 0.1 M HCl for at least 1 h and boil for 10 - 20 min.

3) Wash it thoroughly using MQ water or distilled water and autoclave.

4) Fit a filter in the column using a yellow micropipet tip before use.

Key points to observe:

a. Use a endA1- E. coli strain for plasmid propagation and isolation whenever possible. The instability of plasmids isolated from endA l bacterial strains has been reported .

b. Do not vortex, shake or incubate for more than 5 min in step 3. This may cause shearing of genomic DNA and/or linerization of the supercoiled plasmid. A Iysis time of less than 5 min is important to cause maximum release of plasmid while minimising plasmid denaturation. The lysate should be clear and viscous.

c. Use of cold room or less than 7 min centrifugation may give rise to a dirty supernatant in step 4. If for whatever reason the centrifugation has to be performed at low temperature, the mixture should be transferred to room temperature as quickly as possible after centrifugation.

d. In earlier protocols and in protocols of commercial miniprep plasmid purification kits, less than 1 min centrifugation is recommended to remove ethanol from either the binding resin or a diatomaceous earth column, but we found that under these conditions some ethanol still remained in the diatomaceous earth. Therefore, centrifugation should be at least 3 min in step 7. If necessary, repeat the centrifugation twice. DNA will not be lost.

e. Use only half of the first volume during step 9. If 100 'micro'l is used for the first elution, then we recommend less than 50 'micro'l for the second elution. If the diatomaceous earth is found in the bottom of the tube following centrifugation, transfer the supernatant carefully into a new eppendorf tube.

Troubleshooting and Hints

Very low yields of plasmid - this is usually attributed to a loosely fitting filter in the column. Check whether the filter in the column is fitted lightly. Check the plasmid copy number. Was antibiotic added or not?

Low purity of plasmid with an OD 260/280, greater or less than 1.8 - 2.0. This usually arises from white gelatinous matter remaining above the diatomaceous earth when preparing the solution. Check the diatomaceous earth solution. Check whether endA 1-/ cells were used.

Vacuum is best applied from a steady source such as 'house vacuum'. Syringes tend to stick and give bursts of vacuum.

If the supernatant in step 5 contains cell debris in suspension because of careless transfer, the column will clog. In this case do not discard sample, but scratch column surface slightly with pipette tip to unclog column.

Cheap ICN Practical Grade guanidine hydrochloride is quite suitable, as long as undissolved solids are removed by filtration once the theoretically 6M solution has been made up.

Home made filter columns can be made using microcentrifuge tubes as described by Hansen et al. , but we recommend piercing the bottom of the tube with a needle, from the inside, rather than snipping the bottom off.

In principle it should be possible to scale this up to a macro-prep. We have only worked with 20 ml cultures per prep.

Triton-Prep Method for bacterial DNA Purification

  1. Grow 5 . Harvest in single eppendorf tube .
  2. Resuspend pellet with 300ul STET buffer . After resuspending add 30ul RNase/lysozyme mixture .
  3. Boil for one minute 15 seconds .
  4. Spin in microfuge for at least 15 minutes.
  5. Take supernatant and phenol extract with 150ul STET- saturated phenol.
  6. Spin and take supernatant. Add 1/10 volume 4M lithium chloride . Let sit on ice for 5-10 minutes.
  7. Spin and take supernatant. Add equal volume isopropanol. RT for 5 minutes.
  8. Spin. No pellet will be visible. Don't panic, DNA is stuck to side all the way up tube.
  9. Important: Wash with 80% ethanol
  10. Resuspend pellet in 50-200ul.

Lysozyme/ RNase mixture

10mg/ml lysozyme

1mg/ml RNase

50mM Tris-HCl pH8.0

Store at -20oC in small aliquots. Do not refreeze after thawing.

STET

8% sucrose

5% Triton X-100

50mM Tris-HCl

50mM EDTA pH 8.0

Filter sterilize. Store at 4oC

1) Endotoxin - you must assume that all standard laboratory glassware will be coated with loads of endotoxin. Wherever possible use sterile, specifically endotoxin-free disposable plastics. If you need to use glassware or other materials that are not guaranteed endotoxin free you should soak them in 0.5M NaOH and then rinse thoroughly with endotoxin free water. Likewise, assume any source of water or buffers are contaminated unless you have checked them using the standard LAL assay or similar . Generally speaking, once you have endotoxin in any of your reagents or samples it is alomost impossible to remove it .

Supplies / Equipment

  • sterile 1.5 µl microfuge tubes
  • sterile pipette tips
  • micropipettes
  • waterbath 100°C
  • microfuge 4°C
  • rocking platform 4°C
  • rocking platform 25°C / room temperature

Bacterial Genomic DNA Purification via Qiagen columns

Cultures should be grown in LB for best results. To avoid overloading the Qiagen columns, the correct number of cells/volume of culture should be determined. Qiagen recommends not more than 4x10E9 cells for a mini-column, 2x10E10 for the midi, and 8x10E10 for the maxi. For this is ~0.4-0.8 ml, 2.0-4.0 ml, and 10.0 to 20.0 ml for overnight cultures in LB.

  1. Prepare buffers according to recipe at end. Equilibrate all to room temp. before use.

    Amount/prepMiniMidiMaxiB11 ml3.5 ml11 mlB20.35 ml1.2 ml4 mlQBT2 ml4 ml10 mlQC3 ml15 ml30 mlQF2 ml5 ml15 mlRNaseA0.2 mg0.7 mg2.2 mglysozyme2 mg8 mg30 mgQiagen protease0.9 mg2 mg10 mgor proteinase K

  2. Dissolve Rnase A in buffer B1 to concentration of 200 ug/ml. Stock solutions of lysozyme and proteinase K can be made in dH2O to concentrations of 100 mg/ml and 20 mg/ml respectively.

  3. Pellet cells by centrifugation at 3000-5000x g for 5-10 min. Remove supernatant.

  4. Resuspend the bacterial pellet in 1/3.5/11 ml of Buffer B1 by vortexing at top speed.

  5. Add 20/80/300 ul of lysozyme stock solution and 45/100/500 ul of proteinase K. Incubate at 37 C for at least 30 min.

  6. Add 0.35/1.2/4 ml of Buffer B2 and mix by inversion several times. Incubate at 50 C for 30 min. Mix well; this step is important for efficient deproteinization. It is also important that the lysate becomes clear at this stage.

  7. Equilibrate Qiagen genomic tip with 2x1/4/10 ml Buffer QBT, and allow the tip to empty by gravity flow.

  8. Vortex the lysate for 5-10 sec and apply onto the equilibrated column. Again, allow it to pass through column by gravity flow. Flow can be assisted with gentle positive pressure but it is also OK to dilute the lysate with an equal volume of Buffer QBT prior to loading. The latter is preferable by virtue of personal experience.

  9. Wash the column with 3x1 ml/2x7.5ml/2x15 ml of Buffer QC. Allow buffer to pass through by gravity flow. Two washes should be enough.

  10. Elute the genomic DNA with 2x1 ml/5 ml/15 ml of Buffer QF. Precipitate with 0.7 volumes isopropanol, equilibrated to room temperature. The DNA should be spoolable; if not, pellet the precipitate by centrifugation at 5000 x g. Wash the precipitate with 70% EtOH and dry. Resuspend in the appropriate solvent .

    Solutions:Buffer Composition B150 mM EDTA, 50 mM Tris/HCl, 0.5% Tween 20, 0.5% Triton X-100 B23 M GuHCl, 20% Tween 20 QBT750 mM NaCl, 50 mM MOPS, 15 % ethanol, 0.15% triton X-100, pH 7.0 QC1.0 M NaCl, 50 mM MOPS, 15% ethanol, pH 7.0 QF1.25 M NaCl, 50 mM Tris/HCl, 15 % ethanol, pH 8.5

Chromosomal DNA Extraction from Gram-positive BacteriaThis procedure was originally developed for Listeria monocytogenes but has worked well with other Gram bacteria we've tried.

Pellet cells from 10 ml overnight cultures in BHI or LB and wash in 5 ml of 0.1X SSC. Resuspend in 1 ml 10 mM Tris-HCl containing 20 % sucrose , add lysozyme to 2.5 mg/ml, and incubate at 37 C for 45 min. Add 9 ml lysis buffer , and incubate additional 30 min at 37 C. Phenol and chloroform extract lysed cells, and ethanol precipitate the DNA with 0.1 vol. 3 M sodium acetate, pH 4.8 and 2 vol. 95% ethanol. Spool out DNA with a glass rod, wash once with 80% ethanol before drying. Some bacterial species may require a longer incubation in lysozyme. For Renibacterium salmoninarum , lysozyme incubations overnight at 37 C worked very well with high yields of DNA

2) Concentration - if convenient volumes are being processed you can proceed straight to the ammonium sulphate precipitation step. If you have larger volumes, or your antibody is present at low concentrations/poor yields, we find it useful to concentrate the supernatants by circulating them through the same type of hollow fibre dialysis cartridges we use for cell growth, allowing the slight pressure of circulation to force water out through the membrane, and rapidly concentrating the antibody/proteins up to 100 fold. The advantage of this method is very large volumes can be processed using sterile and endotoxin free components.

Reagents

  • Immunoprecipitation dilution buffer
  • Immunoprecipitation wash buffer
  • Immunoprecipitation buffer
  • Electrophoresis sample buffer
  • Antisera 0.5 - 5 µl, antibody-agarose conjugates
  • 10% SDS

Ammonium Sulphate Precipitation

Procedure 1

  1. Preparation of cell lysate
    1. Rinse the cells on a confluent 60mm culture dish with PBS,
    2. Lyse with the addition of 0.5ml boiling 1% SDS, 1.0mM sodium vanadate, 10mM Tris-HCl pH 7.4.
    3. Transfer lysate to a 1.5ml microcentrifuge tube and boil for an additional 5 minutes.
    4. Sonicate briefly or pass several times through a 26 gauge needle and centrifuge for 5 minutes. This is a "total cell lysate ".
  2. Preparation of cell lysate
    1. Rinse the cells in a confluent 60 mm culture dish with PBS, then lyse the cells with the addition of 0.5 ml cold immunoprecipitation buffer , maintaining constant agitation for 30 minutes at 4°C.
    2. Scrape the cells from the dish and pass several times through a 26 gauge needle to disperse any large aggregates.
    3. Remove insoluble materials by centrifuging the cell lysates for 15 minutes at 4°C in a microcntrifuge. The supernatant is the "total cell lysate ".
  3. Immunoprecipitation with soluble antibodies
    1. To a microcentrifuge tube, add 1-5 µg polyclonal or monoclonal antibody, 400 µl dH2O, 500 µl of 2 x immunoprecipitation buffer, and 100 µl total lysate containing approximately 200 - 500 µg total protein.
    2. Vortex and incubate at 4°C for 1 hour. .
    3. Add 50 µl 10 % protein A or protein A-sepharose. Vortex and incubate with agitation for 30 minutes at 4°C.
    4. Wash 3 x by centrifugation with 1 x immunoprecipitation buffer.
    5. Resuspend the pellet in 30 µl of 2 x concentrated electrophoresis sample buffer, boil for 5 min., the centrifuge for 5 min.
    6. Load the supernatant onto an SDS-PAGE gel and electrophorese normally. Transfer to PVDF and probe with appropriate antibodies.
  4. Immunoprecipitation with Antibody-Agarose Conjugates
    1. To a microcentrifuge tube, add 25µl of a 50% suspension of antibody-agarose conjugate, 400µl H2O, 500µl 2X immunoprecipitation buffer, and 100µl total lysate containing 200-500µg total protein.
    2. Vortex and incubate at 4°C for 1 hour.
    3. Wash with 1X immunoprecipitation buffer by centrifuging for 4 minutes in a microcentrifuge. Repeat wash.
    4. Resuspend the pellet in 30µl 2X concentrated electrophoresis sample buffer, boil for 5 minutes, then centrifuge for 5 minutes.
    5. Load the supernatant onto an SDS-PAGE gel and electrophorese normally. Transfer to PVDF and probe with appropriate antibodies.

Prepare saturated 2SO4, using Analar Grade reagent, 1kg per litre endotoxin free water, and autoclave in NaOH treated glass bottles. Store at 4 deg C. Mix supernatant above excess crystals before use to ensure even saturation.

Procedure 2

Immunoprecipitation of a serum sample

  1. Add 1/10 vol. of 10% SDS to sample. Vortex and heat for 4 min. at 100°C.
  2. Immediately add 4 volumes of immunoprecipitation dilution buffer. Vortex and spin 5 min. in microfuge.
  3. Transfer to a new tube, avoiding pellet.
  4. To a microfuge tube, add 1-5 µg of primary antibody. Place on 4 °C rocker overnight.
  5. If primary antibody was not already conjugated with agarose, add 20 µl protein A-sepharose per tube.
  6. Place on 25°C rocker for 90 min., spin down pellet .
  7. Wash pellet 3 times with detergent wash buffer, then once with non-detergent wash buffer.
  8. Add treatment buffer. Add 1/20 volume 2-mercaptoethanol.
  9. Boil samples 4 min. Spin down pellet. Remove and save supernatant, this is the sample.

Add equal volume of saturated 2SO4 to your antibody sample. Mix well and leave to stand at 4 deg C . Spin out antibody precipitate . Discard supernatant, and redissolve pellet in endotoxin free water. Repeat saturated 2SO4 precipitation.

Time required

Afternoon and next morning.

Redissolve Ig fraction pellet in endotoxin free water and dialyse against PBS using at least 3 changes of 100x volume. .

Finally, centrifuge and 0.2 micron sterile filter to remove any grossly aggregated material, and store frozen at -20 deg C.

If your supernant was produced in a hollow fibre cartridge with good yields then you should expect that more than 90% of the protein visible on a native gel electrophoresis will be the monoclonal antibody. In this case we normally estimate antibody concentration simply by the OD280/1.4 value. This material is suitable for most purposes, including in vitro conjugation to FITC or biotin, or for experimental use in vivo. Levels of endotoxin at this point should be less than 20EU per ml using the LAL assay.

Ion Exchange Purification

This step is necessary if your starting yield was lower than 1mg/ml or if higher purity is required. Ion exchange, rather than affinity purification is recommended, because ion exchange materials are high capacity, cheap enough to be disposable, and can easily be sanitized with 0.5M NaOH. The following method is applicable to most rat and mouse IgG antibodies , although individual monoclonal antibodies may require modification of either the pH or ionic strength for optimal purification. Cation exchange using Fast Flow S-Sepharose at pH5.4 has the advantage that most antibodies will bind, while albumin and degraded antibody will not. This allows reasonable yields in a simple, step-wise absorption, wash, elution method performed entirely in tubes with centrifugation, minimizing the risks of introducing endotoxin by the use of columns etc. However, for maximal purity, and elimination of all residual bovine Ig and transferrin, elution and fractionation with a slowly increasing NaCl concentration would be advantageous.

Dialyse your 2SO4 precipitated antibody preparation into 1x Malonate buffer . At this point you will probably get quite a large precipitate - this is mostly degraded proteins - cellular components, FCS and denatured antibody .

 To make a 10x stock of buffer A:

 Malonic Acid 104g NaOH 60g Betaine 20g Make to 2 litres with endotoxin free water.  should be pH 5.2 - pH 5.4 at 25 deg C   Sterile filter to 0.2micron and store at 4 deg C 

 To make Buffer B : Make 1x buffer A, but containing 0.5M NaCl Filter to 0.2micron if not used immediately

Spin out precipitate and discard. If you dialysed at 4 deg C allow to warm to room temperature. Ensure all steps from here are with room temperature buffers etc.

Measure OD280 and adjust to between 1-20 mg/ml in buffer A. You will need to use the minimum amount of Fast Flow S-Sepharose to maximise yield, so estimate the total amount of Ig in your preparation and use 1ml of packed gel for every 20mg of antibody. If you use excess the eluted antibody will be more dilute.

Prepare Fast Flow S-Sepharose: if this is from a fresh bottle it is usually sufficient just to wash it three times in buffer A. Otherwise you should first wash once in 0.5M NaOH, followed by buffer B, and then three washes with buffer A.

Incubate the antibody previously dialysed into buffer A with correct volume of S-Sepharose for 1 hour at room temperature, with gentle rotation.

Remove supernatant , and wash three times with buffer A .

Elute antibody by adding 1 gel volume of buffer B, rotating at room temperature for 5 mins, centrifuging, and collecting supernatant. Repeat with another column volume and pool eluates.

It is a good idea to make certain that no Sepharose remains that could re-adsorb antibody in the eluate, either by spinning again or passing the antibody through a sterile 0.2 micron filter.

Dialyse against two changes of PBS, check final OD280 and purity on SDS reducing PAGE and native PAGE. Generally, approx. 95% of the protein should be antibody, appearing as a single band on native PAGE. With optimal conditions, yields should exceed 70% and the final Ig concentration should be from 5-15 mg/ml.

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