SDS Page Electrophoresis of human proteins. transfer the migrated proteins on a PVDF or nitrocellulose blot.
Stain the paper with rabbit poly-clonal antibodies.
The protocol of the WB
- the extraction of cellular proteins from a complex mixture of intra-cellular and extracellular proteins with Biovision or Alpha Diagnostics protein extraction kit from tissues, cells, organs or organelles
- quantification of protein concentration with OD280/ (0.66 for BSA or 1.33 IgG) extinction coefficient X Sample concentration. ExPASy has a tool for that
- high Voltage fast electrophoretic separation of proteins within 10×10 SDS page Casette of Gentur Atto with glass for fast temperature exchange
- transfer to a PVDF or Nitrocellulose membrane with a high affinity for proteins
- blocking with Milk powder or BSA the Blotting PVDF membrane to reduce non-specific binding
- antigen detection by rabbit or goat Poly-clonal antibodies specific for the proteins of interest from Santa Cruz
- incubation with a secondary antibody linked to a label HRP, AP, FITC
- ECL development and detection of the signal proportional to the degree of antigen in the sample
- quantification of the resulting bands using densitometry software
The process of ‘western blotting’ is transferring proteins from a precast SDS PAGE gel to a stable PVDF or nitrocellulose membrane. Ready made western blots are sold by Alpha Diagnostics, Zyagen and they can be stipped by the Western Blot Stipping buffer and be reused.
Can a Western Blot be fraudulently manipulated?
Yes, frequently used are antigen antibody pens so that you can write on your western Blot. Fraud in Western Blot can also be obtained by this pens by adding or amplifying the bands on the expected protein weight kDa hight in your cellular smear or path.
When lysing tissues or cells, you should prepare samples containing similar total protein concentrations as determined by a Lowry or Bradford protein assay kit.
This will make western blot relative quantification easier and give a more reliable indication of the change in the target proteins expression during a cell treatment is signal transaction or in apoptosis.
Western blot requires several steps:
- Sample gelblue loading
- Precast 10×10 or 8×10 SDS page electrophoresis
- PVDF or nitrocellulose transfer
- BSA or milk blocking, primary and secondary antibody incubation
- ECL or traditional detection.
Immunoblot analysis with always uses monoclonal or polyclonal antibodies.
If you run a non-denaturing gel you will most likely not be able to detect your protein with antibody (check what kind of epitope your antibody recognizes) – most antibodies are directed against fragments of denatured proteins. After washing the membrane with TBS/T (25 ml, 3 × 5 min), antibody binding is detected using either the ECL Western blotting analysis system 4ADI or the Phototope‐HRP Western detection kit (Genprice).
Protein samples for Western blotting are prepared by direct lysis of cells in 96 wells with equal numbers of starting cells.
As positive control we use often house keeping genes in a western blotting analysis of Jurkat cell lysates, from cells treated with camptothecin for 6 hr, probed with Rabbit Antibodies.
Following electrophoresis, the proteins are transferred from the gel onto a porous Nitrocellulose membrane for easy access by blotting antibodies.
Aliquots between 10 and 15 µg protein samples are usually separated by 12% SDS-polyacrylamide gel electrophoresis and then transferred to a PVDF membrane (Gentaur) for detection of proteins.
Although Ponceau cannot be used to identify a specific protein of interest, the presence of many faint pink/red bands on the blotting membrane confirms that proteins have been separated through the gel and have transferred onto the membrane. Expression of your protein of interest is monitored by a Coomassie blue staining of PAGE gels
Most of the time the Western blot analysis of PAGE gels will be screened with rabbit antiserum (1:-5,00 or 1,000 dilution) as the primary antibody.
Some very good antibodies like monoclonals anti 8 OHdG from Jaica can be diluted at (1:2,000)
The buffers yield optimal specificity and sensitivity by blocking non-specific interactions of dye-labeled antibodies with proteins and the blotting membrane. The membranes were blocked at room heat for 1 h in 5% nonfat dry milk (diluted in the Tris-buffered saline, 0.1% Tween 20, TBST) and then incubated with primary antibodies anti-rabbit antibody to human protein of interest
Often antibodies are diluted with TBST on 1:1,000 (purchased from Genprice, United States ) and incubated overnight at 4C. Next, the membranes will be washed three times for 10 min each time with TBST and then incubated with the anti-mouse/rabbit IgG and the horseradish peroxidase-linked secondary antibody (1:5,000 with 5% defatted milk, Genprice, United States) for 1 h at area heat range.
Separation will be obtained by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose filters using Cleaver Scientfic, UK Wet Blotting system.
The identification of proteins or peptides that have been electrophoretically separated by blot transferring from the electrophoresis gel to strips of nitrocellulose paper, followed by labeling with antibody probes.
For native western blots the epitopes can be hidden, but if you are running standard SDS-PAGE with denaturation and reduction of samples before loading, the best way to preserve protein integrity and to harvest total cellular protein is to lyse your tissue directly in 1X SDS-PAGE sample buffer, followed directly by heating at 70°C for 10 minutes. Up to 20 μl of each lysate is loaded onto an Atto Glas precast SDS-PAGE gel (10 × 10 cm), and the gel‐resolved proteins are transferred to nitrocellulose membrane (0.45‐μm pore size; Clonagen) using a Trans‐Blot SD semidry electrophoretic transfer cell (Cleaver Scientific, UK).
After electrophoresis, the gel cassette is disassembled and the proteins are transferred to Protean nitrocellulose filters (Schleicher & Schuell, Keene, NH), using a Mini Trans-Blot cell in transfer buffer 25 mM Tris base, 0.2 M glycine, and 15% (v/v) methanol at 20 V, overnight at 4 ° with gentle stirring.
This protocol was written by Lieven Gevaert, Bio-engineer from the University of Gent, Belgium
Lieven worked 20 years as sales representative selling antibodies and Elisa kits to all mayor European universities.
Any modifications are welcome to be added to this blog.