Western blotting样品准备 （一）

实验概要

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.

实验步骤

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.