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Western Blot

Preparation of Buffer for WB

2017-06-29Author:adminpraise:0Download

 1.0 mol/L Tris•HClpH6.8                  

  Tris

  15.14 g

  Ultrapure water 

  200 mL

Dissolve the Tris in the ultrapure water, and then add concentrated HCl to adjust the pH to be 6.8. Add ultrapure water until the total volume is 250 mL. Store the prepared buffer at room temperature after high temperature sterilization.

 

1.5 mol/L Tris•HClpH8.8                 

  Tris

  45.43 g

  Ultrapure water

  200 mL

Dissolve the Tris in the ultrapure water, and then add concentrated HCl to adjust the pH to be 8.8. Add ultrapure water until the total volume is 250mL. Store the prepared buffer at room temperature after high temperature sterilization.

 

10% SDS       

Dissolve 10g SDS in 100 mL of distilled water by incubating in 50water bath, and store the prepared buffer at room temperature. If there is any precipitate during the long time storage, the buffer can still be used after dissolution by water bath.

 

10% Ammonium persulfateAPS

Dissolve 0.1g ammonium persulfate in 1.0 mL of ultrapure water, and then store the prepared buffer at 4 for 1 week (-20 for long time storage).

 

30% Acrylamide

  Acrylamide

  29 g

  N,N'-Methylenebisacrylamide

  1 g

Dissolve the acrylamide and N,N'-Methylenebisacrylamide in 50 mL water. Then add distilled water until the total volume is 100 mL. Store the prepared buffer at room temperature in brown bottle.

 

10× PBS (2L)

  NaCl

  160 g

  KCl

  4 g

  Na2HPO4·12H2O

  22.9 g

  KH2PO4

  4 g

  Distilled water

  1 L

Mix all of the substances above homogeneously and then add NaOH to adjust the pH to be 7.4. Add distilled water until the total volume is 2L. Store the buffer at room temperature.

 

Reduced 5×SDS (Loading Buffer)

  0.5 mol/L Tris•HCl (pH6.8)

  2.5 mL

  Dithiothreitol (DTT)

  0.39 g

  SDS

  0.5 g

  Bromophenol Blue

  0.025 g

  Glycerin

  2.5 mL

Mix all of the substances above homogeneously, and then aliquot the buffer and respectively. Store them in 1.5 mL centrifugal tubes at 4 for weeks(-20 for long time storage).

 

Electrophoretic Buffer

  Tris

  3.03g

  Glycine

  18.77g

  SDS

  1g

Add distilled water to dissolve the substances above until the total volume is 1000 mL. Store the buffer at room temperature. The prepared solution can be repeatedly used for 3 to 5 times.

 

Transfer Buffer

1×Transfer Buffer

  Glycine

  11.25 g

  Tris

  2.375 g

  SDS

  0.375 g

  Methanol

  200 mL

Add distilled water to dissolve the substances above until the total volume is 1000 mL. Store the buffer at 4. The prepared solution can be repeatedly used for 3 to 5 times.

 

TBS Buffer

  NaCl

  8.5 g

  Tris

  1.2 g

  SDS

  0.375 g

  Methanol

  200 mL

Add distilled water to dissolve the NaCl and Tris, SDS, and then add glacial acetic acid to adjust the pH to be 7.2. Add 200mL Methanol to the buffer then add distilled water until the total volume is 1000mL. Store the buffer at room temperature.

 

Eluant TBST Solution

Mix 1000 mL of TBS solution with 1 mL of Tween-20, then homogeneously mix the buffer. Prepare the buffer before when needed.

 

Blocking BufferTBST buffer with 5% skimmed milk

  Skimmed milk

  5 g

  TBST

  100 mL

Dissolve the skimmed milk in TBST. The prepared TBST buffer can be used for only one time.

 

RIPA Lysis Buffer, Medium

  Reagent

Amount

  Final concentration

  Tris-base

0.6055 g

  50 mM

  NaCl

0.8766 g

  150 mM

  EDTA·2Na.2H2O

0.037224 g

  1 mM

  SDS

0.1 g

  0.1%

  Sodium deoxycholate

     0.5 g

  0.5%

  NP-40

1 mL

  1%

Dissolve Tris-base, NaCl, EDTA and SDS with distilled water, and then add concentrated HCI to adjust the pH to be 7.4. Add 5 mL of the sodium deoxycholate solution of 10% that is prepared separately in advance (the working concentration of sodium deoxycholate solution is 0.5%), and add 1 mL of NP-40. Mix them and add distilled water until the total volume is 100 mL. Aliquot the buffer and store at -20.

 


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