Westernblotting樣品準備

實驗概要

Preparation of  lysis buffers, protease and phosphatase inhibitors, lysate from cell  culture, lysate from tissues, protein concentration, samples for loading  into gels (denatured and native, reduced and non-reduced) for Western  blotting.

實驗步驟

1. Lysis buffers

To prepare samples for running on a gel, cells and tissues need to be  lysed to release the proteins of interest. This solubilizes the  proteins so they can migrate individually through a separating gel.  There are many recipes for lysis buffers but a few will serve for most  Western blotting experiments. In brief, they differ in their ability to  solubilize proteins, with those containing sodium dodecyl sulfate and  other ionic detergents considered to be the harshest and therefore most  likely to give the highest yield.

Most Abcam antibodies recognise reduced and denatured protein and  should be used under reducing and denaturing conditions. It is important  to note though that some antibodies will only recognize a protein in  its native, non-denatured form and will not recognize a protein that has  been extracted with a denaturing detergent (SDS, deoxycholate, and  somewhat less denaturing, Triton X-100 and NP-40).

The main consideration then when choosing a lysis buffer is whether  the antibody one has chosen will recognize denatured samples. When this  is not the case, it will be noted on the antibody datasheet, and buffers  without detergent or with relatively mild non-ionic detergents (NP-40,  Triton X-100) should be used.

Protein Location And Lysis Buffer Choice

*Proteins that are found exclusively or predominantly in a  sub-cellular location can be enriched in a lysate of the sub-cellular  fraction compared to whole cell or tissue lysates. This can be useful  when trying to obtain a signal for a weakly-expressed protein. For  instance, a nuclear protein will be a larger proportion of the total  protein in a nuclear lysate than it will be in a whole-cell or  whole-tissue lysate, making it possible to load more of the protein per  gel lane. Another advantage is the removal of potentially cross-reactive  proteins present in the unused fractions. Please consult our separate  protocols for sub-cellular fractionation.

    (1) Nonidet-P40 (NP40) buffer

150 mM sodium chloride

1.0% NP-40 (Triton X-100 can be substituted for NP-40)

50 mM Tris, pH 8.0

This is a popular buffer for studying proteins that are cytoplasmic,  or membrane-bound, or for whole cell extracts. If there is concern that  the protein of interest is not being completely extracted from insoluble  material or aggregates, RIPA buffer may be more suitable, as it  contains ionic detergents that may more readily bring the proteins into  solution.

    (2) RIPA buffer (Radio Immuno Precipitation Assay buffer)

150 mM sodium chloride

1.0% NP-40 or Triton X-100

0.5% sodium deoxycholate

0.1% SDS (sodium dodecyl sulphate)

50 mM Tris, pH 8.0

RIPA buffer is also useful for whole cell extracts and membrane-bound  proteins, and may be preferable to NP-40 or Triton X100-only buffers  for extracting nuclear proteins. It will disrupt protein-protein  interactions and may therefore be problematic for  immunoprecipitations/pull down assays.

The 10% sodium deoxycholate stock solution (5 g into 50 ml) must be protected from light.

In cases where it is important to preserve protein-protein  interactions or to minimize denaturation (for example, when it is known  that the antibody to be used will only recognize a non-denatured  epitope), a buffer without ionic detergents (e.g. SDS) and ideally  without non-ionic detergents (e.g. Triton X-100) should be used. Cell  lysis with detergent-free buffer is achieved by mechanical shearing,  often with a Dounce homogenizer or by passing cells through a syringe  tip. In these cases a simple Tris buffer will suffice, but as noted  above, buffers with detergents are required to release membrane- or  cytoskeleton- bound proteins.

    (3) Tris-HCl buffer    

20 mM Tris-HCl, pH 7.5

    (4) Tris-Triton buffer

(Cytoskeletal proteins)

10 mM Tris, pH 7.4

100 mM NaCl

1 mM EDTA

1 mM EGTA

1% Triton X-100

10% glycerol

0.1% SDS

0.5% deoxycholate

All four of these buffers will keep at 4°C for several weeks or for up to a year aliquotted and stored at -20°C.

2. Protease and phosphatase inhibitors

As soon as lysis occurs, proteolysis, dephosphorylation and  denaturation begin. These events can be slowed down tremendously if  samples are kept on ice or at 4°C at all times and appropriate  inhibitors are added fresh to the lysis buffer.

Ready-to-use cocktails of inhibitors from various suppliers are available but you can make your own cocktail.

Sodium orthovanadate preparation

All steps to be performed in a fume hood.

          a. Prepare a 100 mM solution in double distilled water.

          b. Set pH to 9.0 with HCl.

          c. Boil until colorless. Minimize volume change due to evaporation by covering loosely.

          d. Cool to room temperature.

          e. Set pH to 9.0 again.

          f. Boil again until colorless.

          g. Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling.

          h. Bring up to the initial volume with water.

          i. Store in aliquots at - 20°C. Discard if samples turn yellow

3. Preparation of lysate from cell culture

    (1) Place the cell culture dish in ice and wash the cells with ice-cold PBS.

    (2) Drain the PBS, then add ice-cold lysis buffer (1 ml per 10⁷ cells/100mm dish/3750px² flask; 0.5ml per 5x10⁶ cells/60mm dish/1875px² flask).

    (3) Scrape adherent cells off the dish using a cold plastic cell  scraper, then gently transfer the cell suspension into a pre-cooled  microfuge tube.

    (4) Maintain constant agitation for 30 minutes at 4°C.

    (5) Centrifuge in a microcentrifuge at 4°C.

You may have to vary the centrifugation force and time depending on  the cell type; a guideline is 20 minutes at 12,000 rpm but this must be  determined by the end-user (e.g. leukocytes need a very light  centrifugation).

    (6) Gently remove the tubes from the centrifuge and place on  ice, aspirate the supernatant and place in a fresh tube kept on ice, and  discard the pellet.

4. Preparation of lysate from tissues

    (1) Dissect the tissue of interest with clean tools, on ice  preferably, and as quickly as possible to prevent degradation by  proteases.

    (2) Place the tissue in round-bottom microfuge tubes or Eppendorf  tubes and immerse in liquid nitrogen to “snap freeze”. Store samples at  -80°C for later use or keep on ice for immediate homogenization.

For a ~5 mg piece of tissue, add ~300 μl lysis buffer rapidly to the  tube, homogenize with an electric homogenizer, rinse the blade twice  with another 2x300 μl lysis buffer, then maintain constant agitation for  2 hours at 4°C (e.g place on an orbital shaker in the fridge). Volumes  of lysis buffer must be determined in relation to the amount of tissue  present (protein extract should not be too diluted to avoid loss of  protein and large volumes of samples to be loaded onto gels. The minimum  concentration is 0.1 mg/ml, optimal concentration is 1-5 mg/ml).

    (3) Centrifuge for 20 min at 12000 rpm at 4°C in a  microcentrifuge. Gently remove the tubes from the centrifuge and place  on ice, aspirate the supernatant and place in a fresh tube kept on ice;  discard the pellet.

The buffer (with inhibitors) should be ice-cold prior to homogenization.

5. Determination of protein concentration

Perform a Bradford assay, a Lowry assay or a BCA assay. Bovine serum albumin (BSA) is a frequently-used protein standard.

Once you have determined the concentration of each sample, you can  freeze them at -20°C or -80°C for later use or prepare for  immunoprecipitation or for loading onto a gel.

6. Preparation of samples for loading into gels: denatured and native, reduced and non-reduced.

    (1) Denatured, reduced samples

Antibodies typically recognize a small portion of the protein of  interest (referred to as the epitope) and this domain may reside within  the 3D conformation of the protein. To enable access of the antibody to  this portion it is necessary to unfold the protein, i.e. denature it.

To denature, use a loading buffer with the anionic denaturing  detergent sodium dodecyl sulfate (SDS), and boil the mixture at 95-100°C  for 5 minutes. Heating at 70°C for 5-10 minutes is also acceptable and  may be preferable when studying multi-pass membrane proteins. These tend  to aggregate when boiled and the aggregates may not enter the gel  efficiently.

The standard loading buffer is called 2X Laemmli buffer, first  described in Nature, 1970 Aug 15;227(5259):680-5. It can also be made at  4X and 6X strength to minimize dilution of the samples. The 2X is to be  mixed in a 1:1 ratio with the sample.

Laemmli 2X buffer:

4% SDS

10% 2-mercaptoehtanol

20% glycerol

0.004% bromophenol blue

0.125 M Tris HCl

Check the pH and bring it to pH 6.8.

When SDS is used with proteins, all of the proteins become negatively  charged by their attachment to the SDS anions. SDS denatures proteins  by “wrapping around” the polypeptide backbone. SDS binds to proteins  fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a  negative charge to the polypeptide in proportion to its length - i.e.,  the denatured polypeptides become “rods” of negative charge clouds with  equal charge or charge densities per unit length.

In denaturing SDS-PAGE separations, therefore, migration is  determined not by intrinsic electrical charge of the polypeptide, but by  molecular weight. SDS grade is of utmost importance: a protein stained  background along individual gel tracts with indistinct or slightly  distinct protein bands are indicative of old or poor quality SDS.

It is usually necessary to reduce disulphide bridges in proteins  before they adopt the random-coil configuration necessary for separation  by size by using ?-mercaptoethanol or dithiothreitol (DTT).

Glycerol is added to the loading buffer to increase the density of  the sample to be loaded and hence maintain the sample at the bottom of  the well, restricting overflow and uneven gel loading.

To enable visualization of the migration of proteins it is common to  include in the loading buffer a small anionic dye molecule (e.g.,  bromophenol blue). Since the dye is anionic and small, it will migrate  the fastest of any component in the mixture to be separated and provide a  migration front to monitor the separation progress.

During protein sample treatment the sample should be mixed by vortexing before and after the heating step for best resolution.

    (2) Native and non-reduced samples

Alternatively, an antibody may recognize an epitope made up of  non-contiguous amino acids. Although the amino acids of the epitope are  separated from one another in the primary sequence, they are close to  each other in the folded three-dimensional structure of the protein, and  the antibody will only recognize the epitope as it exists on the the  surface of the folded structure.

It is imperative in these circumstances to run a Western Blot in  non-denaturing conditions, and this will be noted on the datasheet in  the applications section. In general, a non-denaturing condition simply  means leaving SDS out of the sample and migration buffers and not  heating the samples.

Certain antibodies only recognize protein in its non-reduced form  i.e. in an oxidized form (particularly on cysteine residues) and the  reducing agents ?-mercaptoethanol and DTT must be left out of the  loading buffer and migration buffer (non reducing conditions).

Rule of thumb: Reduce and denature unless the datasheet specifies otherwise.