Buffers

From Ruzin, 1999. Plant Microtechnique and Microscopy

The following are recipes for a number of common biological buffers taken from Ruzin, 1999 Plant Microtechnique and Microscopy. When choosing one for a particular application select a buffer based on its pH optimum and biological properties rather than its historical use. Many buffer species have an impact on biological systems, enzyme activities, substrates, or cofactors (Perrin and Dempsey, 1974). For example, phosphate buffers inhibit the activity of several metabolic enzymes including carboxylase, fumarase, and phosphoglucomutase. Barbiturate uncouples oxidative phosphorylation. Tris buffer reacts with primary amines and modifies electron transport and phosphorylation in chloroplasts. Tris also inhibits respiratory enzymes in mitochondria. HEPES does not have these negative effects yet buffers at a similar pH range. MOPS and MES decompose when autoclaved in the presence of glucose. Keep buffer concentration as low as possible yet enough to maintain pH.

GLYCINE–HCL; PH 2.2–3.6, PKA = 2.35
Combine 25 ml 0.2 M glycine and x ml HCl and dilute to 100 ml with DI (Dawson, et al., 1969).
x (ml) pH
22.0 2.2
16.2 2.4
12.1 2.6
8.4 2.8
5.7 3.0
4.1 3.2
3.2 3.4
2.5 3.6

SODIUM ACETATE; PH 3.6–5.6, PKA = 4.76

Combine the following proportions of 0.1N acetic acid and 0.1N sodium acetate (Pearse, 1980).
acetic acid sodium acetate pH
185 15 3.6
176 24 3.8
164 36 4.0
147 53 4.2
126 74 4.4
102 98 4.6
80 120 4.8
59 141 5.0
42 158 5.2
29 171 5.4
19 181 5.6

BUFFERED SALINE (PBS, TBS, TNT, PBT)

Buffered saline solutions are used frequently when performing immunolocalization experiments. There are many variations. Presented here are three common formulations (Mishkind, et al., 1987).

PBS 20x stock TBS
Potassium chloride 4 g 53.6 mM Potassium chloride 4 g
NaCl 160 g 274 mM NaCl 160 g
Potassium phosphate monobasic 4 g 29.4 mM Tris buffer (10 mM, pH 7.5) to 1 liter
Sodium phosphate dibasic (7•H2O) DI 43.2 g 17.5 mM to 1 liter Use TBS when performing immunocytochemical
experiments on phosphate-sensitive tissues
(photosynthetic tissues typically)
TNT PBT
NaCl 150 mM PBS to vol
Tris buffer (100 mM, pH 7.5) to 1 liter Tween 20 1% (v/v)

CACODYLATE BUFFER; PH 5.0–7.4, PKA = 6.27

Sodium cacodylate buffer [Na(CH3)2 AsO2 • 3H2O] is a alternative to Sørensen’s phosphate buffer. It has good pH buffering capacity within the range of pH 5.0–7.4. Cacodylate was introduced for electron microscopy applications by Sabatini et al. (1962) as a method of avoiding adding additional phosphates to sample preparations. Mitochondria and other organelles can be damaged when exposed to the high concentrations of phosphates present in Sørensen’s buffers. Also, cacodylate will not react with aldehyde fixatives as will amine-containing buffers (e.g., Tris). Its efficacy in fixation solutions may be a result of the metabolism-inhibiting effect of the arsenate rather than any special buffering capacity.
Prepare a 0.2 M stock solution of sodium cacodylate in water (4.28 g/100 ml). Add the following amounts of 0.2 M HCl per 100 ml cacodylate stock solution, followed by the addition of DI to a final volume of 400 ml, to obtain 0.05 M cacodylate buffer at the desired pH (Dawes, 1971).

0.2 M HCl pH
94.0 5.0
90.0 5.2
86.0 5.4
78.4 5.6
69.6 5.8
59.2 6.0
47.6 6.2
36.6 6.4
26.6 6.6
18.6 6.8
12.6 7.0
8.4 7.2
5.4 7.4

“GOOD” BUFFERS; PKA = 6.15–8.06

Tris–HCl (pKa = 8.06) and maleate (pKa = 6.26) have a working range of pH 5.0–8.6 and may be used successfully to buffer staining solutions (e.g., Toluidine Blue O). Avoid Tris with aldehyde fixatives or osmium tetroxide, however, as the aldehydes reacts with the amino group of Tris, resulting in the loss of buffering capacity. PIPES (pKa = 6.80) is commonly used as a buffer for retention of actin filaments during fixation. Other useful biological buffers include HEPES (pKa = 7.55), MES (pKa = 6.15), and MOPS (pKa = 7.20) (Good, et al., 1966; Perrin and Dempsey, 1974).

CITRATE BUFFER; PH 3.0–6.2, PKA = 6.40 Citrate buffer (Gomori, 1955) stock solutions: A: 0.1 M citric acid; B: 0.1 M sodium citrate. Use x ml A + y ml B and dilute to 100 ml with 50 ml DI.

0.1 M citric acid 0.1 M sodium citrate pH
46.5 3.5 3.0
43.7 6.3 3.2
40.0 10.0 3.4
37.0 13.0 3.6
35.0 15.0 3.8
33.0 17.0 4.0
31.5 18.5 4.2
28.0 22.0 4.4
25.5 24.5 4.6
23.0 27.0 4.8
20.5 29.5 5.0
18.0 32.0 5.2
16.0 34.0 5.4
13.7 36.3 5.6
11.8 38.2 5.8
9.5 41.5 6.0
7.2 42.8 6.2

SØRENSEN’S PHOSPHATE BUFFER; PH 5.8–8.0, PKA = 7.20

Mix appropriate volumes of stock and add an equal volume of distilled water to make a final 0.1 M Sørensen’s phosphate buffer solution (Sørensen, 1909; Gomori, 1955). Keep in mind that high levels of phosphate may be somewhat toxic to plant cells (Sabatini, et al., 1962) and thus Sørensen’s buffer may not be appropriate for some experiments.
Stock solutions: A 0.2 M NaH2PO4 B 0.2 M Na2HPO4
A (ml) B (ml) pH
92.0 8.0 5.8
87.7 12.3 6.0
81.5 18.5 6.2
68.5 31.5 6.5
62.5 37.5 6.6
56.5 43.5 6.7
51.0 49.0 6.8
45.0 55.0 6.9
39.0 61.0 7.0
33.0 67.0 7.1
28.0 72.0 7.2
23.0 77.0 7.3
19.0 81.0 7.4
16.0 84.0 7.5
8.5 91.5 7.8
5.3 94.7 8.0

PHOSPHATE–CITRATE BUFFER; PH 2.2–8.0, PKA = 7.20/6.40

Add the following to create 100 ml of phosphate/citrate buffer solution. Stock solutions are
0.2 M dibasic sodium phosphate; 0.1 M citric acid (Pearse, 1980).
0.2 M Na2HPO4 (ml) 0.1 M citrate (ml) pH
5.4 44.6 2.6
7.8 42.2 2.8
10.2 39.8 3.0
12.3 37.7 3.2
14.1 35.9 3.4
16.1 33.9 3.6
17.7 32.3 3.8
19.3 30.7 4.0
20.6 29.4 4.2
22.2 27.8 4.4
23.3 26.7 4.6
24.8 25.2 4.8
25.7 24.3 5.0
26.7 23.3 5.2
27.8 22.2 5.4
29.0 21.0 5.6
30.3 19.7 5.8
32.1 17.9 6.0
33.1 16.9 6.2
34.6 15.4 6.4
36.4 13.6 6.6
40.9 9.1 6.8
43.6 6.5 7.0

BARBITAL BUFFER; PH 6.8–9.2, PKA = 7.98

Add the following to create 200 ml of buffered solution. To 50 ml of 0.2 M sodium barbital (Veronal,
41.2 g in 1000 ml) add x ml 0.2 M HCl to create the buffered solution and dilute to 200 ml with DI (Gomori, 1955).
0.2 M HCl (ml) pH
1.5 9.2
2.5 9.0
4.0 8.8
6.0 8.6
9.0 8.4
12.7 8.2
17.5 8.0
22.5 7.8
27.5 7.6
32.5 7.4
39.0 7.2
43.0 7.0
45.0 6.8

TRIS BUFFERS

Tris buffers are used commonly in microtechnique applications involving molecular biological procedures. Listed here are a number of common Tris formulations (Maniatis, et al., 1982).
Solution Preparation
Tris, 1 M stock Tris base DI Dissolve and adjust pH with the following approximate amount of HCl: pH 7.4 pH 7.6 pH 8.0 121.1 g 800 ml 70 ml 60 ml 42 ml
EDTA, 0.5 M Disodium ethylene diamine tetraacetate Adjust pH to approx. 8.0 and stir until dissolved 186.1 g
SSC, 20x NaCl NaCitrate DI Adjust pH to 7.0 with NaOH then add DI to 1 liter 175.3 g 88.2 g 800 ml
SSPE, 20x NaCl NaH2PO4 • H2O EDTA DI Adjust pH to 7.4 with NaOH then add DI to 1 liter 174 g 27.6 g 7.4 g 800 ml
TE Tris EDTA Adjust pH using Tris stock solution 10 mM 1 mM
STE (TNE) Tris NaCl EDTA Adjust pH to 8.0 using Tris stock solution 10 mM 100 mM 1 mM

GLYCINE– NAOH BUFFER; PH 8.6–10.6, PKA = 9.78

Stock solutions:
0.2 M glycine
0.2 NaOH
Combine 25 ml glycine stock solution with x ml 0.2 M NaOH and dilute with DI to make a 100 ml solution (Pearse, 1980).
0.2 M NaOH pH
2.0 8.6
3.0 8.8
4.4 9.0
6.0 9.2
8.4 9.4
11.2 9.6
13.6 9.8
19.3 10.4
22.75 10.6