This webpage was created as an assignment for an undergraduate course at Davidson College.

The Western Blot

The Western blot, commonly called the protein blot, is a method used to separate proteins by molecular weight (MW in Da or kDa). Similar to the Southern blot for DNA and the Northern blot for RNA, the Western blot utilizes separation of the studied molecule through a medium. Among its various uses in the labratory, the Western blot has recently become a notable molecular tool because of its role in the ELISA test for detection of HIV. The most common Western blot method uses sodium dodecyl sulfate as a denaturing agent and polyacrylamide gel electrophoresis as the medium for separation (SDS-PAGE). Like Southern and Northern blotting, Western blotting shares several fundamental steps: extraction, denaturation, separation, transfer, blocking, hybridization with a probe, washing, viewing and analysis.

Extraction:

total protein extraction is performed from studied tissue culture
once isolated, the protein can be denatured

Denaturation:

the protein is denatured with sodium dodecyl sulfate (SDS), an anionic surfactant that binds strongly to the protein
SDS reduces the protein to primary linear structure and coats it with a negative charge
SDS is typically applied in the ratio, 1.4 g SDS : 1 g protein

Separation:

the typical medium for Western blotting is polyacrylamide gel electrophoresis
SDS-PAGE is advantageous:
- polyacrylamide produces high resolution separations (as little as 1 ng protein can be detected) - 2 runs through polyacrylamide are sufficient for the most difficult separations
- polyacrylamide allows for both soluble and insoluble protein samples
- polyacrylamide allows for confirmation via Ab/antigen probing
a gel of 10-15% is used to run proteins
a buffer solution is used to support the negatively-charged proteins, and the gel is submerged in this buffer solution
the proteins and MW standard are loaded into the gel
a voltage is applied to the electrophoresis system
the negatively-charged, denatured proteins run through the gel from negative to positive electrode (see Figure 1 below)

Figure 1. Depiction of SDS-PAGE setup utilizing a method that combines separation and transfer. (Image - Short Protocols in Molecular Biology, Second Edition, p. 10-34)

Transfer:

once run, the gel is transferred to a nitrocellulose membrane, which freezes the positions of the proteins
transfer can be completed by simple capillary action of a stack of paper towels on top of the gel (see Figure 2 below)
other membranes that can be used:
- PVDF (polyvinylidene diflouride)- less fragile than nitrocellulose and is commonly used when proteins are to be sequenced
- nylon
- diazo-modified cellulose - advantageous because of its high level of protein binding

Figure 2. Nitrocellulose transfer via capillary action. (Highly sophisticated paper towel method). Permission pending from source.

Blocking:

the purpose of blocking is to coat the membrane with excess protein prevent the probe from binding to the unsaturated membrane itself
a solution of excess proteins is applied to the membrane
during this period, the blot may also be stained if desired - typical stains include: Ponceau S, Amido Black, and Coomassie Brilliant Blue

Hybridization:

the probe for a Western blot is an antibody that binds specifically to the studied protein's epitope
the Ab probe may be synthesized to include:
- isotope 125I for viewing via radioautography
- alkaline phosphotase conjugated ligand or horseradish peroxidase conjugated ligand for viewing via enzymatic detection
- secondary antibody that includes chemiluminescent characteristics for viewing by fluorescence
the membrane is coated in buffer containing the Ab probe and allowed to bind for a period of hours
after the protein/Ab hybrids have been formed, the membrane may be washed

Washing:

the membrane is washed several times with buffer to release any unbound, excess Ab probe

Viewing:

the method of protein detection for the Western blot varies as does the type of Ab probe used
for 125I probe, the membrane is exposed to X-Ray and the film is developed
for enzymatic probe, substrate is added to membrane and either colored product is formed or light is produced and exposes film, which is then developed (see Figure 4 below)
for chemiluminescent probe, location is depicted via exposure and subsequent fluorescence

Figure 3. Depicts detection by enzymatic Ab/protein hybrid via method developed by Amersham.
As the membrane is treated with peracid, the secondary Ab probe reacts enzymatically and produces light as a byproduct. The light produced is detected by film, which is then exposed for analysis. (Image - Basic Protein and Peptide Protocols, Vol 32 , p.235)

Figure 4. Top image: Film exposed after staining with SYPRO Ruby protein blot stain. Bottom image: Film after enzymatic action and exposure. Permission pending from source.

Analysis:

from a good SDS-PAGE Western blot, one can determine:
- relative MW in Da or kDa of one protein from another
- accurate MW of one protein if a sufficient MW standard is run

The Resolving Power of SDS-PAGE:

Figure 5. The resolving accuracy of MW in a typical SDS-PAGE Western blot. Note the complex MW standard in lane 8. Permission pending from source.

Useful Links:

See a real protocol for the Western Blot by Kirkegaard and Perry Laboratories here.

Buy materials to run your own Western blot here.

Try a common Western blot analysis problem here.

References:

Ausubel, Frederick M. et. al., Short Protocols in Molecular Biology Second Edition. John Wiley and Sons: New York, 1997.

p. 10-33 - 10-35.

Walker, John M. ed. Basic Protein and Peptide Protocols Vol. 32, Humana Press: Totowa, NY, 1994. 189, 209-221.

White, Brian. Southerns, Northerns, Westerns, & Cloning: Molecular Searching Techniques.1995. February 17, 2003

< http://web.mit.edu/esgbio/www/rdna/rdna.html#electrophoresis >.

Campbell, Malcolm. Western Blot. 2001. February 16, 2003

< http://bio.davidson.edu/Courses/genomics/method/Westernblot.html >.

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Send comments, questions, and suggestions to: Charles Wood