The buffers for Western blotting

The buffers for Western blotting [1]

Transfer buffer for SDS-PAGs, pH 8.3 (for the proteins <80 000 Da)

25 mM Tris base (Fw 121.1 g/mol) 3.03 g

192 mM Glycine (Fw 75.07 g/mol) 14.4 g

10 MeOH* 100 ml

with or without SDS**

add H₂O to1000 ml. Do NOT adjust the pH of Transfer buffer!!!

Transfer buffer should always be stored at +4C, such improving heat dissipation during the transfer. Reuse of the buffer is not advised.

* - Methanol (10-20) improves binding of SDS proteins to nitrocellulose membranes ONLY, and might cause transfer problems for the proteins with higher molecular weight (> 100 kDa). PVDF does not require MeOH in the transfer buffer.

** - SDS (0.025-0.1) aids in eluting the proteins from the gel, but it may reduce the binding efficiency of those proteins to the nitrocellulose membrane, and may increase the binding efficiency of the proteins to PVDF membrane.

Generally Tris buffers allow more efficient transfer than acetate or phosphate buffers.

Tris buffered saline (TBS), pH 7.4

20 mM Tris/HCl (Fw 121.1 g/mol) 2.42 g

150 mM NaCl (Fw 58.44 g/mol) 8.8 g

add H₂O to1000 ml.

The buffer should be stable for at least 3 months if stored at room temperature.

TBS-Tween and TBS-Triton, pH 7.4

Dilute the required amount of Tween 20 or Triton X-100 in the corresponding buffer just before the use. The 0.1 Tween 20 (1 ml/1000 ml) and 0.2-0.5 Triton X-100 (2-5 ml/1000 ml) are suitable for most blotting applications.

 

Stripping buffer, pH 2.2

200 mM Glycine (Fw 75.07 g/mol) 15 g

0.1 SDS 1 g

1 Tween-20 10 ml

add

10 mM DTT 1.542 g

or

5-mercaptoethanol 50 ml

add H₂O to1000 ml.

Incubate the plot for 1 hr at room temperature. After incubation, if antibodies have to be saved for next experiment, addjust the pH of solution with Tris to 7.4, add 50 glycerol, and store at –20-70C. Otherwise, discard the solution, wash the membrane with TBS and incubate with other antibodies.

 

2004 03 27 – Oliver & Julius

The buffers for Western blotting [2]

Basic proteins in Tris, glycine and MeOH buffer at pH 8.3 may assume a state near isoelectric neutrality and thus transfer poorly. Therefore, it is advisable to use buffers with pH of 9.2 to 10.5.

Transfer buffer for SDS-PAGs, pH 9.2 (for the proteins 20 000-400 000 Da)

48 mM Tris base (Fw 121.1 g/mol) 5.82 g

39 mM Glycine (Fw 75.07 g/mol) 2.93 g

10 MeOH 100 ml

with or without SDS

add H₂O to1000 ml. Do NOT adjust the pH of Transfer buffer!!!

 

CAPS-transfer buffer

(for N-terminal sequencing of SDS-PAGE separated proteins)

20 mM CAPS (Fw 221.3 g/mol) 4.42 g in 800 ml H₂O

adjust pH with NaOH to 9.5-10.5 and then add:

10 MeOH 100 ml

sodium thioglycolate (reductant) 11 mg

and H₂O to1000 ml. Use the buffer only once!!!

(1) Phosphate buffered saline (PBS), pH 7.4

58 mM Na₂HPO₄ (anhydrous; Fw 142 g/mol) 8.24 g

17 mM NaH₂PO₄-H₂O (Fw 137.99 g/mol) 2.35 g

68 mM NaCl (Fw 58.44 g/mol) 3.98 g

add H₂O to1000 ml and check pH.

(2) Phosphate buffered saline (PBS), pH 7.5

80 mM Na₂HPO₄ (anhydrous; Fw 142 g/mol) 11.5 g

20 mM NaH₂PO₄-H₂O (Fw 137.99 g/mol) 2.96 g

100 mM NaCl (Fw 58.44 g/mol) 5.84 g

add H₂O to1000 ml and check pH.

(3) 0.1 M Phosphate buffered saline (PBS)

Prepare two stock solutions:

0.2 M Na₂HPO₄ (anhydrous; Fw 142 g/mol) 28.4 g/L

0.2 M NaH₂PO₄-H₂O (Fw 137.99 g/mol) 27.6 g/L

Start with a known quantity of stock dibasic solution and add stock monobasic until the desirable pH is reached. Adding more dibasic will raise the pH, and monobasic-will lover it. Dilute the solution with water by half to obtain 0.1 M buffer and add 0.9 g of NaCl per 100 ml of buffer (0.9 final; pH drops by 0.2 units!).

The buffers should be stable for up to 3 months if stored in a fridge.

Important! Almost every laboratory has its own recipes for TBS and PBS buffers and they differ in the composition, concentration of the reagents, and pH. Choose the best!

 

2003 07 18 – Oliver & Julius