Western Blotting

Western Blot Protocol

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Summary Workflow For Western Blot Protocol

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Electrophoresis

(A) Gel Preparation

The first step of gel preparation is to determine the gel percentage based on the molecular weight of your protein sample:

Protein Size (kDa) >100 30-100 10-30 <10
Gel Percentage 8% 10% 12% 15%

If you are not sure of the size of your protein or are looking at proteins of a variety of molecular weights, then a gradient gel may provide the best resolution.

    Notes
  • We recommend using the SDS-PAGE Gel Preparation Kit available from us (Boster Catalog # AR0138). It contains most of the reagents for the gel preparation and can be used to make both SDS-PAGE gel and non-native PAGE gel.
  • Many protocols are available for gel preparation. Please refer to the manufacturer’s guidelines for use of specific products.
  • Pre-cast gels may also be used instead of making your own gel.

(I) Resolving Gel Preparation

  1. Determine the volume needed and gently mix the ingredients for the chosen percentage of the resolving gel. Make sure to blend the solution gently to avoid too much oxygen from entering the solution.
  2. Pour the gel solution slowly into your gel casting form.
  3. Layer the top of the gel with distilled water to prevent oxygen from entering the gel.
  4. Incubate at 37°C for 30-60 minutes for the gel solution to fully polymerize until it becomes solid.
  5. Remove the water from the polymerized resolving gel (absorb excess with paper towel).

(II) Stacking Gel Preparation

  1. Determine the volume needed, gently mix the ingredients, and absorb water on the solid resolving gel with filter.
  2. Slowly pour the stacking gel solution on top of the running gel.
  3. Carefully insert the sample comb to avoid bubbles. There should be no bubbles between the comb teeth.
  4. Incubate at 37°C for 30-60 minutes. When the gel solution is fully polymerized and solid, gently take out the comb.

(B) Pre-electrophoresis Sample Preparation

  • Mix the extracted protein sample with 4X Dual Color Protein Loading Buffer (Boster Catalog # AR1142) at 3:1 ratio (i.e. add 300µg sample to 100µL loading buffer).

    Dual Color Protein Loading Buffer is designed to prevent protein degradation during sample heating prior to electrophoresis and is able to work against pH changes during SDS-PAGE run. Many proteins are sensitive to pH changes that result from temperature fluctuations of Tris buffers during electrophoresis. It contains two tracking dyes: blue (Bromophenol Blue) for tracking the progress of electrophoresis and pink (Pyronin Y) for monitoring protein transfer to the membrane. Refer to the datasheet on our website for more information.

  • You may also use one of the following reagents/methods instead of the Dual Color Protein Loading Buffer:
    • SDS-PAGE Protein Loading Buffer 2X (Reducing) (Boster Catalog # AR0131) at 1:1 ratio (i.e. add 100µg sample to 100µL loading buffer)
    • SDS-PAGE Protein Loading Buffer 5X (Reducing) (Boster Catalog # AR1112) at 4:1 ratio (i.e. add 400µg sample to 100µL loading buffer)
    • Tricine SDS Sample Buffer 2X (Boster Catalog # AR1143) at 1:1 ratio (i.e. add 100µg sample to 100µL loading buffer) if the protein with low molecular weight (<10KD)
    • Laemmli 2X Buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris-HCl; pH 6.8) at 1:1 ratio (i.e. add 100µg sample to 100µL loading buffer)
  • Denature the sample/loading buffer mixture in a 100°C water bath for 5 minutes (or follow the manufacturer instructions). Alternatively, the mixture can be stored in aliquots at -20°C for several months or at 4°C for 1-2 weeks before use.

(C) Loading Samples & Running Electrophoresis

  1. Place the gel in the electrophoresis apparatus.
  2. Fill both buffer chambers with SDS-PAGE Electrophoresis Buffer (25 mMTris base, 190 mM glycine and 0.1% SDS; pH 8.3). We recommend using our buffer (Boster Catalog # AR0139). Fill both buffer chambers with SDS-PAGE Electrophoresis Buffer (25 mMTris base, 190 mM glycine and 0.1% SDS; pH 8.3) to a level above the sample loading well between the two-layer glass pane. Ensure the bottom of the gel is immersed in the loading buffer and the liquid level inside the two-layer glass pane is higher than the outside level. We recommend using our buffer (Boster Catalog # AR0139).
  3. Carefully remove the well-creating comb from the gel and rinse the wells with the electrophoresis buffer.
  4. Pipette your samples into the wells quickly to prevent possible sample diffusion inside the well. As an example, for a well that can contain maximum 30 µL, load 20 to 25 µL of 1 µg/µL sample per well.
  5. Pipette 10 µL of appropriate controls and/or molecular weight standards in separate well(s). The loaded samples should include the treated sample, tissue and cell sample protein for positive control, recombinant protein for positive control, and the marker.
  6. Properly connect the anode and cathode of the electrophoresis.
  7. Turn on the power to run electrophoresis at 100-130V* until the bromophenol blue dye reaches the gel bottom (this can take 1.5 to 3 hours). You should observe fine bubbles from the gel apparatus bottom as this observation indicates that sufficient electric current is generated.
  8. Turn off the power when the protein samples have finished migrating in the gel.
    Notes:
  • To prevent sample diffusion, try to shorten the amount of time it takes to load samples as much as possible.
  • In a discontinuous system, the electrophoresis voltage for stacking gel (70-80V) is lower than that for resolving gel (90-110V) to ensure that proteins are concentrated on the same level before running into the resolving gel.
  • *The applied voltage should be adjusted according to the gel thickness, power supply used, and resolution desired.

Protein Transfer (To Membrane)

(A) Gel Staining (Optional)

After electrophoresis, we recommend using one of our gel staining solutions to determine if the electrophoretic separation worked. Please refer to the datasheet(s) on our website for more information.

  • Coomassie Blue Staining & Destaining Solution (Boster Catalog # AR0140)
  • Coomassie Blue Fast Staining Solution (Boster Catalog # AR0170)
  • Silver Stain Kit (Boster Catalog # AR0171)

Note: Stained gel cannot be used in the subsequent protein transfer procedure.

(B) Wet Transfer

  1. Blotting Membrane Preparation
    • Cut the blotting membrane (NC or PVDF) according to the size of your gel (Tips: Cut a good supply of membranes in advance! Store in a cool, dry place).
    • Carefully mark the membrane orientation by cutting a corner or marking it with a pencil.
    • Soak the membrane in methanol for 1 minute.
    • Immerse the membrane for 5 minutes in 1X transfer buffer (25 mMTris base, 190 mM glycine and 20% methanol; pH 8.3) (Boster Catalog # AR1149). Rock the membrane gently until it sinks and water no longer beads up on the surface.
western blot cathode anode
  • Transfer Cassette
    • Take out gel and immerse the gel in transfer buffer (Boster Catalog # AR1149) for 15-30 minutes.
    • Based on a sandwich model, install the electric transfer cassette in the following order:
      Foam Pad (Sponge) → Filter Paper (Transfer Pad) → Gel → NC Membrane→ Filter Paper (Transfer Pad) → Foam Pad (Sponge)
      Make sure the "sandwich" is clamped tightly, so no air bubbles are within it. Ensure the gel is closest to the cathode and the membrane is closest to the anode (please see the image below). The negatively charged amino acid and protein will migrate towards the anode.
      We recommend using one of the filter papers from Boster:
      • Western Blotting Filter Paper, 0.158mm thick, 12.5cm×12.5cm (Boster Catalog # AR0172)
      • Western Blotting Filter Paper, 0.158mm thick, 9cm×7.5cm (Boster Catalog # AR0173)
        • Soak two filter papers in a separate container with the same transfer buffer
        • Crack open the transfer cassette with a spatula and make sure to loosen the cassette hold all the way around before carefully pulling apart the two halves (Before doing anything with the gel, such as cutting it, pay careful attention to the location of lane #1.).
        • Cut the gel according to the size of the membrane with a razor blade and then cut the corner on the side of the gel with lane #1.
        • Immerse the gel in 1X transfer buffer for 15-30 minutes.
        • Place the gray or black plate of the transfer cassette on a clean surface.
        • Place one pre-wetted foam pad on the gray side of cassette.
        • Place a moistened sheet of filter paper on the foam pad.
        • Carefully peel the gel off of the remaining half of the gel cassette and place it onto the filter paper (Moisten the gel with transfer buffer and use a serologically clean pipette or a Falcon tube, as if it were a rolling pin, to roll air bubbles out of the membrane).
        • Place the membrane onto the gel with the corners match up. Once the membrane contacts the gel, it should not be moved or “ghost bands” can result.
        • Complete the sandwich by placing a piece of filter paper onto the membrane.
        • Add the second foam pad on top of the filter paper.
        • Lock the transfer cassette firmly with the white latch. Be careful not to move the gel and filter paper sandwich. Make sure the foam pads, filter papers, and membrane are thoroughly immersed in the transfer buffer.
  • (iii) Protein Transfer Run
    1. Fill transfer tank with an adequate amount of 1X transfer buffer.
    2. Firmly insert transfer cassette into the slot of the transfer apparatus.
    3. Place the lid on top of the transfer tank and make sure the electrodes are lined correctly. The gel should be closer to the cathode and the membrane should be closer to the anode. Negatively charged proteins will migrate towards the anode.
    4. Set power source to constant voltage and operate at 25V for 30 minutes*.
    5. Check the protein transfer efficiency by membrane staining: Place NC membrane in Ponceau S staining (0.2% w/v Ponceau S; 5% glacial acetic acid) or our Ponceau S Solution (Boster Catalog # AR0142) for 5-10 minutes. A visible red band will appear. The membrane may be de-stained completely by repeatedly washing in wash buffer.
  • western blot protein transfer
      Notes
    • The transfer can be completed overnight at a lower voltage (e.g. 10V).
    • Constant current of 150-300mA is usually applicable. Transfer time and voltage should be optimized according to the gel concentration. Higher gel concentration requires additional time.

    Membrane Blocking

    • Rinse the blotting membrane 3 times using TBS Wash Buffer (20 mMTris, pH 7.5; 150 mMNaCl; 0.05% Tween 20) (Boster Catalog # AR0144) at room temperature for 10 minutes each time.
    • After rinsing, immerse the blotting membrane in TBS Blocking Buffer (5% non-fat dry milk in buffer of 20 mMTris, pH 7.5; 150 mMNaCl) (Boster Catalog # AR0143) and incubate for 1.5-2 hours at room temperature (or overnight 4°C) with shaking. Alternatively, buffer containing non-fat dried milk, gelatin, or BSA can be used. For use with biotin systems or detection of phosphoproteins, non-fat dried milk is not recommended.

    Antibody Incubation

    After blocking, the membrane is incubated with a primary antibody (that binds to the target protein) followed by an enzyme conjugated secondary antibody (HRP- or AP-conjugated secondary antibody).

    • Dilute the primary antibody with the Antibody Diluent Buffer (Boster Catalog # AR1106-2). Follow the antibody protocol from the manufacturer for optimal dilution.
    • Incubate the primary antibody and the membrane at 4°C overnight or for 1-2 hours at room temperature. For the best results, incubation time and antibody concentration may need to be optimized.
    • Wash the membrane 3 times with the TBS Wash Buffer for 10 minutes each to remove unbound antibody.
    • Dilute the secondary antibody with the TBS Blocking Buffer (Boster Catalog # AR0143). Follow the antibody protocol from the manufacturer for optimal dilution.
    • Incubate the secondary antibody and the membrane at 4°C overnight or 1-2 hours at room temperature on a shaker.
    • Wash the membrane 3-6 times with the TBS Wash Buffer for 10 minutes each to remove unbound antibody.

    Signal Detection

    The choice of western blot antibody detection methods depends on specific substances that are labeled or bound to the secondary antibody. ECL chemiluminiscent detection system and DAB chromogenic detection system are typically used in western blotting.

    In this section, we provide the protocols for the Enhanced Chemiluminescence Detection (ECL) and colorimetric detection (DAB) methods. Use the method that fits your preferences and criteria.

    (A) Enhanced Chemiluminescence Detection (ECL)

    Boster ECL chemiluminescent system depends on incubation of the western blot with a substrate that will luminesce when exposed to HRP on the secondary antibody. The light is then detected and captured by photographic film.

    1. ECL Substrate Preparation
      • Choose the correct ECL kit† according to the species that the primary antibody is raised.
        Origin of Primary Antibody Species Catalog # of ECL Kit*
        Mouse IgG EK1001
        Rabbit IgG EK1002
        Goat IgG EK1003
        Rat IgG EK1004
        Mouse IgM EK1005

        *Each kit has sufficient reagents for 800 cm2 of membrane.

        †Instead of using the ECL kit which provides 1) chromogenic reagents A and B (20X concentrated; 5 mL), 2) blocking buffer and 3) HRP-conjugated secondary antibody, one may use one of the following standalone chromogenic reagents A and B from Boster:

        Reagent A Reagent B
        Product Conc. Volume Conc. Volume Catalog #
        ECL Western Blotting Substrate 1X** 100 mL 1X** 100 mL AR1170
        Hypersensitive WB Chemiluminescent Substrate 20X 5 mL 20X 5 mL CR0001-5
        Hypersensitive WB Chemiluminescent Substrate 20X 10 mL 20X 10 mL CR0001-10
        Hypersensitive WB Chemiluminescent Substrate 20X 25 mL 20X 25 mL CR0001-25

        ** Ready-to-use

      • Prepare the ECL substrate solution by mixing the following and use the solution within two hours of preparation:
        • 50 µL of 20X Chromogenic Reagent A (Luminol & Luminious Enhancer)
        • 50 µL of 20X Chromogenic Reagent B (Peroxidase & Stabilizer)
        • 1 mL of distilled water
    2. Membrane Treatment
      • Thoroughly cover the membrane with the substrate solution (use 1 mL of solution for 10 cm2 of membrane).
      • Incubate the membrane at room temperature until bands appear (usually 1-5 minutes; incubation time can be estimated in dark room).
      • Gently blot the edge of the membrane on a piece of paper to remove excess any substrate solution.
      • Put a clear preservative film or transparent glass paper over the membrane and remove any air bubbles observed.
    3. Film Development and Fixing

      Record the test result using autoradiography film or CHEMIDOC in a darkroom. For autoradiography film, exposure time for the specific antigen depends on the developing effect, which could range from seconds to minutes.

      Using WB Developing Fixing Kit (Boster Catalog # AR0132), develop and fix the film in a dark room immediately. Alternatively, fluorescence CCD scan, digital imager, or luminometer can be used.

      • Put the X-ray film over the membrane.
      • Develop the film by immersing it in developing solution for 10 seconds to 10 minutes. Determine the exposure time required by observing under red light and stop developing once the film achieves the experimental purpose. Multiple exposures may be necessary for the optimal signal to noise ratio.
      • Wash the film with clean water (to remove the developing solution completely) and stop washing when bands appear.
      • Immerse the film in fixing solution for 3-5 minutes.
      • Wash the film with clean water to remove the fixing solution.
        Notes
      • WB Stripping Buffer (Boster Catalog # AR0153) is recommended to remove primary and secondary antibodies on the membrane if proteins on the membrane need to be reused.
      • Use the control protein levels to normalize the target protein levels.

    (B) Colorimetric Detection

    Prepare DAB or BCIP/NBT substrate solution described below.

    1. DAB Substrate Preparation (For HRP-conjugated secondary antibodies)
      • Choose the correct DAB kit according to the species that the primary antibody is raised and the desirable color:
        Origin of Primary Antibody Species Color Catalog # of DAB Kit
        Mouse IgG Yellow SA2020
        Goat IgG Yellow SA2021
        Rabbit IgG Yellow SA2022
        Rat IgG Yellow SA2023
        Mouse IgG Blue SA2024
        Rabbit IgG Blue SA2025
      • Prepare the DAB substrate solution by mixing the following:
        • 50 µL of 40X Chromogenic Reagent A (DAB)
        • 50 µL of 40X Chromogenic Reagent B (H2O2)
        • 50 µL of 40X Chromogenic Reagent C (TBS Wash Buffer)
        • 2 mL of distilled water
    2. BCIP/NBT Substrate Preparation (For AP-conjugated secondary antibodies)
      • Prepare the BCIP/NBT substrate solution by mixing the following:
        • 50 µL of 20X Chromogenic Reagent A (BCIP/NBT)
        • 50 µL of 20X Chromogenic Reagent B (Tris concentrated buffer, pH 9.4)
        • 1 mL of distilled water
    3. Membrane Treatment
      • Thoroughly cover the membrane with the substrate solution (use 1 mL of solution for 10 cm2 of membrane).
      • Incubate the membrane at room temperature until bands appear (usually 10-30 minutes); incubation for BCIP/NBT should be done in dark.
      • Wash the membrane in distilled water to stop the reaction.
      • Observe the bands and take pictures.