Western blot buffers and stock solutions

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​​​Lysis buffers

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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 7.51 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

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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 waterpH to 7.6 with 12 N HClAdd 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 10×900 mL distilled water1 mL Tween 20

Medium stripping buffer

15 g glycine1 g SDS10 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 M67.5 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 MgCl210 mM KCl0.5 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 mLMgCl2: 1 M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 mLKCl: 1 M = 74.5 g/L, therefore 10 mM = 0.187 g/250 mLDTT: 1 M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 mLNP-40: 0.05%

Nuclear fractionation protocol reagents buffer B

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5 mM HEPES1.5 mM MgCl2​0.2 mM EDTA0.5 mM DTT26% 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 mLMgCl2: 1 M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 mLEDTA: 1 M = 372.2 g/L, therefore 0.2 mM = 0.0186 g/250 mLDTT: 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-1001 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 antibody10 mg BSA900 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)-biotin980 µL TBS pH 7.6-7.8

Bicarbonate/carbonate coating buffer (100 mM)

3.03 g Na2CO36.0 g NaHCO​3 (1 L distilled water) pH 9.6​PBS: 1.16 g Na2HPO40.1 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.

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