Western Blot Sample Preparation Protocol

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Abstract:          Western blot sample preparation plays an important role in obtaining a satisfied western blot band. Protein extraction is the simplest extraction technology.
Keywords:        Western Blot Sample Preparation, Protein Extraction for Western Blot, Western Blotting Techniques, Western Blot Analysis

1. Protein Extraction for Western Blot

1.1. Data Collection

Sample type, amount and the purpose of western blot should be confirmed first. The research on target protein includes the expression level, location and unique properties.

For example:

  • Mouse liver tissue research: The expression level of target protein is very high. The protein expression can be validated by extracting the total protein of the tissue sample.
  • 293 cell research: The target protein is expressed in the cytoplasm and nucleus. Target protein expression level in different components can be compared by separating the cytoplasm and nucleus of the cell sample. Then, cytoplasmic and nucleolus proteins are detected respectively.

1.2. Protein Extraction Protocols

The protein extraction is based on the full protein profile. Furthermore, The protein should be maximally extracted by the proper ratio.

There are various protein extraction reagents in the market. However, their basic components are nearly cell lysates. Protease and phosphatase inhibitors assist to extract proteins in tissues, cells or subcellular fractions. Two kinds of inhibitors prevent the target protein from degradation. PMSF suits for common proteins. Phosphatase inhibitor should be added for validating phosphorylated protein.

During extracting tissue proteins, the position of organ tissues should be noticed. E.g. renal tissue consists of cortex and medullar. The brain is also divided into different regions, belonging to non-homogeneous organs. The protein expression level probably varies in different regions. Thus, the protein should be extracted in the specific region, following your experimental purpose.

2. Western Blot Sample Preparation

2.1. Cell Lysates for Western Blot

To efficiently extract protein, 100-200μl extraction reagent is usually instilled into each well of 6-well plate. 1-2ml extraction reagent is sufficient for per 100 mg tissue.

Before extraction, using PBS to wash cell samples twice to reduce the interference of culture medium on samples. Suspension cell can be centrifuged at 500Xg and then collected for protein extraction. Proteins in adherent cell can be directly extracted in the cultivating container. The trypsin digestion technique is not suggested to use for collecting cells in membrane expressed proteins.

2.2. Tissue Preparation for Western Blot

Take the digestive system related tissues(e.g. stomach, large and small intestine, liver, pancreas etc) and macrophage rich tissue first. Then, take reproductive related tissues (e.g. ovary, uterus, testis etc). Finally, take organs like heart, spleen, kidney, brain etc. If the taken tissues are not used at once, they should be cryopreserved in liquid nitrogen or refrigerator at -80℃. Before extraction, using PBS to wash the blood to prevent the influence of IgG in the blood on the following experiment.

Tissue grinding usually requires for homogenization. Soft tissues can be processed by a tissue grinder which consists of stainless steel or glass hammer and a glass tube. The surface of the hammer and tube wall are used for rough surface treatment. Tight fibrous tissue(e.g. muscle tissue) requires for strong grinding. Solid tissue frozen by liquid nitrogen can be ground by ceramic or stone bowl and frozen rod. The rotatable homogenizer with blades or mixer is especially suitable for large, bony or fibrous samples.

During sample preparation, ultrasonic processing is also suggested for homogenized samples. The extraction should be conducted in ice bath and centrifuged at 12000rpm for 10min at 4℃.

3. Sample Concentration and Storage

3.1. Western Blot Protein Concentration Detection

At the end of protein extraction, BCA method is suggested to detect concentration and control the loading amount.

3.2. Boiling Samples for Western Blot

After the measurement of concentration, the sample concentration should be adjusted to the same value. The sample with the same volume and quality is loaded by mixing with loading buffer, boiling and centrifugation.

3.3. Internal Control Western Blot

Although many methods are applied in the accuracy and concentration of protein extraction, internal control is still required for the strictness of western blot.

4. FineTest Antibodies

FineTest offers over 10,000 Rabbit polyclonal & Mouse monoclonal primary antibodies and 100+ tagged secondary antibodies (e.g. HRP, FITC, Biotin etc). FineTest antibody products are widely applied in ELISA, WB and IHC etc. More than 1,000 antibodies are developed annually.

Some Cited Antibodies:

REFERENCES

[1] Western blotting: sample preparation to detection, PMID: 21189462.
[2] The design of a quantitative western blot experiment, PMID: 24738055.
[3] The necessity of and strategies for improving confidence in the accuracy of western blots, PMID: 25059473.