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Western blot coomassie blue stain

Western blot buffers and stock solutions

Related

  • Western blot resources
    • Western blot protocol
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          • Transfer and staining
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                                      ​​Recipes for western blot buffers and stock solutions.   

                                      Print our buffer and stock solutions.



                                      • Lysis buffers
                                      • Cytoskeletal bound protein extract buffer
                                      • Soluble protein buffer
                                      • Loading, running, transfer, and blocking buffers
                                      • Sodium orthovanadate preparation
                                      • TBS 10x (concentrated Tris-buffered saline)
                                      • TBS 10x alternative recipe (concentrated Tris-buffered saline)
                                      • TBST (Tris-buffered saline, 0.1% Tween 20)
                                      • Medium stripping buffer
                                      • Harsh stripping buffer
                                      • Nuclear fractionation protocol reagents buffer A
                                      • Nuclear fractionation protocol reagents buffer B
                                      • TBS 0.025% Triton X-100
                                      • 1.6% H2O2 (hydrogen peroxide) in TBS
                                      • Primary antibody made up in TBS with 1% BSA
                                      • ABC (avidin-biotin complex) in TBS
                                      • Bicarbonate/carbonate coating buffer (100 mM)

                                      ​​

                                      ​​​Lysis buffers

                                      These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.

                                      RIPA buffer (radioimmunoprecipitation assay buffer)

                                      RIPA buffer contains the ionic detergent sodium deoxycholate as an active constituent and is particularly useful for nuclear membrane disruption for nuclear extracts. A RIPA buffer gives low background but can denature kinases. It can also disrupt protein-protein interactions and may, therefore, be problematic for immunoprecipitations and pull-down assays.

                                      • 50 mM Tris-HCl, pH 8.0
                                      • 150 mM NaCl
                                      • 1% IGEPAL CA-630
                                      • 0.5% sodium deoxycholate
                                      • ​0.1% SDS
                                      • Protease inhibitors

                                      ​The 10% sodium deoxycholate stock solution (5 g into 50 mL) must be protected from light.


                                      NP-40 buffer 

                                      • 150 mM NaCl
                                      • 1.0% NP-40 (possible to substitute with 0.1% Triton X-100)
                                      • 50 mM Tris-HCl, pH 8.0
                                      • Protease inhibitors

                                      ​​Tris-HCl

                                      •  20mM Tris-HCl
                                      • Protease inhibitors


                                      Cytoskeletal bound proteins extract buffer

                                      • 10 mM Tris, pH 7.4
                                      • 100 mM NaCl
                                      • 1 mM EDTA
                                      • 1 mM EGTA
                                      • 1 mM NaF
                                      • ​20 mM Na4P2O7
                                      • 2 mM Na3VO4
                                      • 1% Triton X-100
                                      • 10% glycerol
                                      • 0.1% SDS
                                      • 0.5% deoxycholate


                                      Soluble protein buffer

                                      20 mM Tris-HCl, pH 7.5
                                      1 mM EGTA (Ca2+​ chelator)


                                      ​Loading, running, transfer, and blocking buffers

                                      Loading buffer/Laemmli 2X buffer

                                      • 4% SDS
                                      • 10% 2-mercaptoethanol
                                      • 20% glycerol
                                      • 0.004% bromophenol blue
                                      • 0.125 M Tris-HCl
                                      • Check the pH and adjust it to 6.8

                                      Running buffer (Tris-Glycine/SDS)

                                      • 25 mM Tris base
                                      • 190 mM glycine
                                      •  0.1% SDS
                                      • Check the pH and adjust to 8.3

                                      Transfer buffer (wet)

                                      • 25 mM Tris base
                                      • 190 mM glycine
                                      •  20% methanol
                                      • Check the pH and adjust to 8.3
                                      • For proteins >80 kDa, we recommend including SDS at a final concentration of 0.1%.

                                      Transfer buffer (semi-dry)

                                      • 48 mM Tris
                                      • 39 mM glycine
                                      • 20% methanol
                                      • 0.04% SDS

                                      Blocking buffer

                                      • 3–5% milk or BSA (bovine serum albumin)
                                      • Add to the TBST buffer. Mix well and filter. Failure to filter can lead to spotting, where tiny dark grains will contaminate the blot during color development.


                                      Sodium orthovanadate preparation

                                      All procedures must be carried out under the fume hood.

                                      1. Prepare a 100 mM sodium orthovanadate solution with double distilled water
                                      2. Set pH to 9.0 with HCl
                                      3. Boil until colorless
                                      4. Cool to room temperature
                                      5. Set pH to 9.0 again
                                      6. Boil again until colorless
                                      7. Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling
                                      8. Bring up to the initial volume with water
                                      9. Store in aliquots at -20°C
                                      10. Discard if the samples turn yellow


                                      Avoid large changes in volume during boiling; put a loose lid on the container to protect from evaporation.



                                      TBS 10x (concentrated Tris-buffered saline)

                                      For 1 L:
                                      24 g Tris base (formula weight: 121.1 g)
                                      88 g NaCl (formula weight: 58.4 g)
                                      Dissolve in 900 mL distilled water
                                      pH to 7.6 with 12 N HCl
                                      Add distilled water to a final volume of 1 L

                                      For a 1x solution, mix 1 part of the 10x solution with 9 parts distilled water and adjust pH to 7.6 again. The final molar concentrations of the 1x solution are 20 mM Tris and 150 mM NaCl. 

                                      An alternative recipe for Tris buffer combines Tris base and Tris-HCl. This avoids the large volume of potentially hazardous hydrochloric acid that is needed to neutralize a solution of Tris base alone.



                                      TBS 10x alternative recipe (concentrated Tris-buffered saline)

                                      For 1 L:
                                      24 g Tris-HCl (formula weight: 157.6 g)
                                      5.6 g Tris base (formula weight: 121.1 g)
                                      88 g NaCl (formula weight: 58.4 g)
                                      Dissolve in 900 mL distilled water

                                      1. The pH of the solution should be about 7.6 at room temperature. If too basic, adjust to pH 7.6 with concentrated HCl, and if too acidic, adjust with concentrated NaOH.
                                      2. Add distilled water to a final volume of 1 L.
                                      3. For a 1x solution, mix 1 part 10x with 9 parts distilled water and pH to 7.6 again.
                                      4. The final molar concentrations of the 1x solution are 20 mM Tris and 150 mM NaCl.



                                      TBST (Tris-buffered saline, 0.1% Tween 20)

                                      For 1 L:
                                      100 mL of TBS 10x
                                      900 mL distilled water
                                      1 mL Tween 20



                                      Medium stripping buffer

                                      15 g glycine
                                      1 g SDS
                                      10 mL Tween 20

                                      1. Adjust the volume to 800 mL with ultra pure water.
                                      2. Adjust pH to 2.2.
                                      3. Bring volume up to 1 L with distilled water.



                                      Harsh stripping buffer

                                      This needs to be done under a fume hood.

                                      For 100 mL:
                                      20 mL SDS 10%
                                      12.5 mL Tris HCl, pH 6.8, 0.5 M
                                      67.5 mL distilled water
                                      ​Add 0.8 mL β-mercaptoethanol under the fume hood



                                      Nuclear fractionation protocol reagents buffer A

                                      10 mM HEPES
                                      ​1.5 mM MgCl2
                                      10 mM KCl
                                      0.5 DTT
                                      ​0.05% NP-40 (or 0.05% Igepal or Tergitol) pH 7.9

                                      To prepare 250 mL stock of buffer A:
                                      HEPES: 1 M = 238.3 g/L, therefore 10 mM = 0.59 g/250 mL
                                      MgCl2: 1 M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 mL
                                      KCl: 1 M = 74.5 g/L, therefore 10 mM = 0.187 g/250 mL
                                      DTT: 1 M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 mL
                                      NP-40: 0.05%



                                      Nuclear fractionation protocol reagents buffer B

                                      5 mM HEPES
                                      1.5 mM MgCl2
                                      ​0.2 mM EDTA
                                      0.5 mM DTT
                                      26% glycerol (v/v) pH 7.9

                                      To prepare 250 mL stock of buffer B:
                                      HEPES: 1 M = 238.3 g/L, therefore 5 mM = 0.295 g/250 mL
                                      MgCl2: 1 M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 mL
                                      EDTA: 1 M = 372.2 g/L, therefore 0.2 mM = 0.0186 g/250 mL
                                      DTT: 1 M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 mL
                                      ​26% glycerol (v/v) = 65 mL



                                      TBS 0.025% Triton X-100

                                      For 1 L:
                                      250 µL Triton X-100
                                      1 L TBS pH 7.6–7.8



                                      1.6% H2O2 (hydrogen peroxide) in TBS

                                      For 400 mL:
                                      6.4 mL H2O2 (GPR = 30% w/w)
                                      393.6 mL TBS pH 7.6–7.8



                                      Primary antibody made up in TBS with 1% BSA

                                      Example is of primary antibody used at a dilution of 1:10.

                                      For 1 mL:
                                      ​100 µL primary antibody
                                      10 mg BSA
                                      900 µL TBS pH 7.6–7.8


                                      ABC (avidin-biotin complex) in TBS

                                      Example is of ABC, each part used at a dilution of 1:100.

                                      For 1 mL:
                                      10 µL Streptavidin
                                      ​10 µL HRP (or AP)-biotin
                                      980 µL TBS pH 7.6–7.8



                                      Bicarbonate/carbonate coating buffer (100 mM)

                                      3.03 g Na2CO3
                                      6.0 g NaHCO​3 (1 L distilled water) pH 9.6
                                      ​PBS: 1.16 g Na2HPO4
                                      0.1 g KCl
                                      ​0.1 g K3PO4
                                      ​4 g NaCl (500 mL distilled water) pH 7.4


                                      Protocols are provided by Abcam “AS-IS” based on experimentation in Abcam’s labs using Abcam’s reagents and products; your results from using protocols outside of these conditions may vary.


                                      ​

                                      • Return to our western blot protocols.
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