How Is Western Blot Done?

The following steps are necessary to set up the process on the SDS PAGE gel.

  1. First build the SDS-PAGE gel in conformity with the molecular weight (MW) of the target protein, whether one or many. Recipes may be available if required.
  2. The samples are placed in microfuge tubes and 4X SDS sample buffer is added to bring the total protein amount to 30 – 50 ug per sample. The protein amount should be that obtained by a Bradford or BCA assay for protein.
  3. The microfuge tube samples are mixed by flicking the tubes, after which the tubes are spun for a short time. They are then heated up to 95 - 100℃ for 5 minutes.
  4. The electrophoresis equipment is now assembled and immersed fully in 1X running buffer. The gel combs are then taken out. Any stacking gel remaining in the wells is purged by pipetting each well with running buffer up and down, with the gel-loading tip, as shown in Figure 1.
  5. The samples are placed into the gel along with the corresponding protein markers using a tip.
  6. The gel tank is closed using the lid and the electrophoresis system is turned on using a power pack. The voltage is first set to low while the sample passes through the stacking gel. Once the dye front arrives at the separating layer, the voltage is increased to 120 V or so. Once the dye front has reached the right position the gel running is arrested.

Note: Tris-tricine gels are better when proteins of low MW are to be separated (below 20 kDa) compared to Tris-glycine gels.

Membrane Transfer

It is important to note here that the use of PVDF membranes is highly advisable when dealing with PSQ membranes that have micropores of less than 0.22 microns pore size to deal with protein targets below 30 kDa in size.

  1. The membranes must be soaked in methanol for 30 seconds and then placed into the transfer buffer.
  2. The filter papers and sponges used are soaked in the transfer buffer.
  3. The transfer components are put together in sequence, as shown in Figure 2, making sure that there are no bubbles between any layers. The manufacturer of the blotting equipment will instruct on the choice of semi-dry or wet transfer systems.

Immunoblotting

  1. Once the transfer is complete, it must be washed with distilled water two times.
  2. The bands of the MW ladder should be lightly marked on the membrane in pencil.
  3. If it is required, the membrane can be stained using Ponceau red solution (commercially obtained) for 30 seconds, in order for protein bands to become visible. This should then be washed away using plenty of 1xTBST.
  4. The sample is then blocked using 1xTBST to which 2-5% non-fat dry milk has been added, or 1-5% BSA if phosphorus-containing epitopes are to be detected, and the sample is rocked constantly for an hour or overnight at 4°C.
  5. The primary antibody is now diluted to an initial dilution ratio of 1:1000 using blocking solution; of course, the optimal dilution must be found by trial and error using a dilution series. The membrane is then incubated with primary antibody for an hour or overnight at 4°C with continuous rocking on a benchtop rocking device.
  6. The membrane is washed with 1xTBST three times, at ten minutes each time.
  7. The membrane is incubated using the right HRP-conjugated secondary antibody specific for the host species from which the primary antibody was raised, that has been diluted as per the protocol. It is then incubated with rocking for an hour.
  8. The membrane is then washed three times using 1xTBST in cycles of 10 minutes.
  9. The membrane should not dry out during any stage of this whole blotting technique.

Detecting the Signal

  1. The ECL substrate is prepared in compliance to the manual.
  2. The membrane is incubated with the substrate fully for 1-5 minutes depending on the sensitivity of the ECL substrates; for instance, SuperSignal West Femto Chemiluminescent Substrate (Pierce) is more sensitive.
  3. The membrane is either exposed in a darkroom to an autoradiography film or read off with a chemiluminescence imaging platform.
  4. The film is developed and lined up in proper orientation to the blot, and the MW band ladder is marked straight on to the film. Some details should be added, such as lane content, duration of film exposure and ECL properties.

The best exposure time must be found by using multiple periods of exposure. Fluorescent markers can be used, and in addition the top right corner of the film may be clipped to help ascertain the proper orientation in relation to the blot.

References

About Proteintech Group, IncProteintech Group, Inc

Proteintech: The Benchmark in Antibodies since 2001

Proteintech are a global biotech company and a renowned center of excellence for the manufacture and supply of quality antibodies, ELISA kits and proteins to the life science research community. With offices in the US (Chicago), UK (Manchester) and China (Wuhan) Proteintech are always available to support your research.

Part of Proteintechs early vision was to make all its own products, to the highest standards possible and to take complete responsibility for the quality. With an emphasis on developing antibodies from whole proteins, Proteintech provides researchers with unmatched reliability and reproducibility.


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Last updated: Mar 29, 2019 at 7:33 AM

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