WB Technical Resources

Protocols, optimization tips,
troubleshooting guides,
and more for Western Blot.

Troubleshooting guides

Troubleshooting guides

Download troubleshooting
handbooks for IHC, Western
blot and ELISA for FREE.

Summary Workflow for WBProtocol

Western blot protocol summary
  1. Electrophoresis

        (A) Gel Preparation

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

        Protein Size (kDa) >200 15-200 10-70 12-45 4-40
        Gel Percentage 6% 8% 10% 12% 20%

        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 is 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, respectively.
        • 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

        • Determine volume needed and gently mix the ingredients for the chosen percentage of the resolving gel.
        • Pour the solution into your gel casting form.
        • Layer the top of the gel with distilled water.
        • Wait approx. 30 min for the gel to polymerize completely.
        • Remove the water from the polymerized resolving gel (absorb excess with paper towel).

        (ii) Stacking Gel Preparation

        • Determine volume needed, gently mix the ingredients and pour the stacking gel on top of the running gel.
        • Insert sample comb (avoid bubbles).
        • Allow 30 to 60 min for complete gel polymerization.
      • (B) Pre-electrophoresis Sample Preparation

        • Mix the extracted protein sample with 4X Dual Color Protein Loading Buffer (Boster Catalog # AR1142)at3:1 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 workagainst 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 of 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:
          - 2X SDS-PAGE Protein Loading Buffer (Boster Catalog # AR0131) at 1:1 ratio (i.e. add 100µg sample to 100µLLoading Buffer)
          - 5X SDS-PAGE Protein Loading Buffer (Boster Catalog # AR1112) at 4:1 ratio (i.e. add 400µg sample to 100µL Loading Buffer)
          - 2X SDS-PAGE TricineProtein Loading Buffer (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 buffermixture in a 100°C water bath for 5 min(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 and Running Electrophoresis

        • Place the gel in the electrophoresis apparatus.
        • 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).
        • Carefully remove the well-creating comb from the gel and rinsewells with the electrophoresisbuffer.
        • Pipet your samples into the wellsquickly to prevent possible sample diffusion inside the well (For a well with maximum 30 µL, load 20 to 25 µL of 1 µg/µL sample per well).
        • Pipette 10 µL of appropriate controls and/or molecular weight standards in separate well(s)
        • Connect the anode and cathode of the electrophoresis appropriately.
        • Turn on the power to run electrophoresis at 100V/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 indicated sufficient electric current is generated.
        • Turn off the power when the protein samples have finished migrating in the gel.

        Notes:

        • In a discontinuous system, the electrophoresis voltage for stacking gel is lower than that for resolving gel 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.
  2. 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 works. Refer to the datasheet(s) on our website for more information.

    • Coomassie Blue Staining &DestainingSolutions (Boster Catalog # AR0140)
    • Coomassie Blue Fast Staining Solution (Boster Catalog # AR0170)
    • Protein Silver Staining Reagents (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 min.
      • Immerse the membrane in 5 min with 1X transfer buffer (25 mMTris base, 190 mM glycine and 20% methanol; pH 8.3)[Boster Catalog # AR1149] and rockthe membrane gently until it sinks and water no longer beads up on the surface.
    2. Transfer Cassette
      • Based on a sandwich model, install the electric transfer cassette in the following order:
        Foam Pad → Filter Paper → Gel → Membrane→ Filter Paper→ Foam Pad
        We recommend using one of the transfer pads from Boster:
        • • Fast and Efficient Transfer Pad, 12.5 cm X 12.5 cm (Boster Catalog # AR0172)
        • • Fast and Efficient Transfer Pad, 9 cm X 7.5 cm (Boster Catalog # AR0173) western blot cathode anode
          • -      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 min.
          • -      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 bufferand 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 padon 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 and make sure that thefoam pads, filter papers and membrane are thoroughly immersed in the transfer buffer).
    3. (iii) Protein Transfer Run western blot protein transfer
      • Fill transfer tank with an adequate amount of 1X transfer buffer.
      • Firmly insert transfer cassette into the slot of the transfer apparatus.
      • Place the lid on top of the transfer tank and make sure the electrodes are lined correctly (Gel should be closer to the cathode; membrane should be closer to the anode. Negatively charged proteins will migrate towards the anode).
      • Set power source to constant voltage and operate at 25 V for 30 min*.
      • Check the protein transfer efficiency by membrane staining: Place membrane in Ponceau S staining (0.2% w/v Ponceau S; 5% glacial acetic acid) or our Imprinted Membrane Fast Reversible Protein Staining Reagent (Boster Catalog # AR0142) for 5-10 min (A visible red band will appear; The membrane may be de-stained completely by repeatedly washing in wash buffer).

    Notes

    • The transfer can be completed overnight at a lower voltage, for example, 10 V.
    • Transfer time and voltage should be optimized according to the gel concentration. Higher gel concentration requires additional time.
  3. Membrane Blocking

    • Rinse the blotting membrane 3X using TBS Wash Buffer(20 mMTris, pH 7.5; 150 mMNaCl; 0.05% Tween 20) [Boster Catalog # AR0144] at room temperature for 10 min 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.
  4. Antibody Incubation

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

    • Dilute the primary antibody with the TBS Wash Buffer (Boster Catalog # AR0144); 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 3X with the TBS Wash Buffer for 10 min 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 3X with the TBS Wash Buffer for 10 min each to remove unbound antibody.
  5. Signal Detection

    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)

    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 #
        Hypersensitive ECL Substrate 1X** 100 mL 1X** 100 mL AR1170
        Hypersensitive WB Substrate 20X 5 mL 20X 5 mL CR0001-5
        Hypersensitive WB Substrate 20X 10 mL 20X 10 mL CR0001-10
        Hypersensitive WB 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 min; 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

      Develop and fix the film in a dark room immediately using our recommended WB Developing and Fixing Kit (Boster Catalog # AR0132); 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 secto 10 min (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 forthe 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 min.
      • 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 min); 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.