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Western Blot

Preparation of Buffer for WB

2017-06-29Author:adminpraise:0Download

1.0 mol/L Tris•HClpH6.8

Tris

 30.28 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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