You are on page 1of 5

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. GLYCINEHCL; PH 2.23.6, 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 2.2 22.0 2.4 16.2 2.6 12.1 2.8 8.4 3.0 5.7 3.2 4.1 3.4 3.2 3.6 2.5 SODIUM ACETATE; PH 3.65.6, PKA = 4.76 Combine the following proportions of 0.1N acetic acid and 0.1N sodium acetate (Pearse, 1980). acetic acid sodium pH acetate 15 3.6 185 24 3.8 176 36 4.0 164 53 4.2 147 74 4.4 126 98 4.6 102 120 4.8 80 141 5.0 59 158 5.2 42 171 5.4 29 181 5.6 19 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 Potassium chloride NaCl Potassium phosphate monobasic Sodium phosphate dibasic (7H2O) DI TBS 4g 53.6 mM Potassium chloride 4 g 160 g 274 mM NaCl 160 g 4g 29.4 mM Tris buffer (10 mM, pH 7.5) to 1 liter 43.2 g 17.5 mM Use TBS when performing to 1 liter immunocytochemical

experiments on phosphate-sensitive tissues (photosynthetic tissues typically) TNT NaCl Tris buffer (100 mM, pH 7.5) 150 mM to 1 liter PBS to vol Tween 20 1% (v/v) PBT

CACODYLATE BUFFER; PH 5.07.4, PKA = 6.27 Sodium cacodylate buffer [Na(CH3)2 AsO2 3H2O] is a alternative to Srensens phosphate buffer. It has good pH buffering capacity within the range of pH 5.07.4. 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 Srensens 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 94.0 90.0 86.0 78.4 69.6 59.2 47.6 36.6 26.6 18.6 12.6 8.4 5.4 GOOD BUFFERS; PKA = 6.158.06 TrisHCl (pKa = 8.06) and maleate (pKa = 6.26) have a working range of pH 5.08.6 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 3.06.2, 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 46.5 43.7 40.0 37.0 35.0 33.0 31.5 28.0 0.1 M sodium citrate 3.5 6.3 10.0 13.0 15.0 17.0 18.5 22.0 pH 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 pH 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4

25.5 23.0 20.5 18.0 16.0 13.7 11.8 9.5 7.2

24.5 27.0 29.5 32.0 34.0 36.3 38.2 41.5 42.8

4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2

SRENSENS PHOSPHATE BUFFER; PH 5.88.0, PKA = 7.20 Mix appropriate volumes of stock and add an equal volume of distilled water to make a final 0.1 M Srensens phosphate buffer solution (Srensen, 1909; Gomori, 1955). Keep in mind that high levels of phosphate may be somewhat toxic to plant cells (Sabatini, et al., 1962) and thus Srensens 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 8.0 5.8 92.0 12.3 6.0 87.7 18.5 6.2 81.5 31.5 6.5 68.5 37.5 6.6 62.5 43.5 6.7 56.5 49.0 6.8 51.0 55.0 6.9 45.0 61.0 7.0 39.0 67.0 7.1 33.0 72.0 7.2 28.0 77.0 7.3 23.0 81.0 7.4 19.0 84.0 7.5 16.0 91.5 7.8 8.5 94.7 8.0 5.3 PHOSPHATECITRATE BUFFER; PH 2.28.0, 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 0.1 M citrate pH Na2HPO4 (ml) (ml) 44.6 2.6 5.4 42.2 2.8 7.8 39.8 3.0 10.2 37.7 3.2 12.3 35.9 3.4 14.1 33.9 3.6 16.1 32.3 3.8 17.7 30.7 4.0 19.3 29.4 4.2 20.6 27.8 4.4 22.2 26.7 4.6 23.3

24.8 25.7 26.7 27.8 29.0 30.3 32.1 33.1 34.6 36.4 40.9 43.6 BARBITAL BUFFER; PH 6.89.2, PKA = 7.98

25.2 24.3 23.3 22.2 21.0 19.7 17.9 16.9 15.4 13.6 9.1 6.5

4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0

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 9.2 1.5 9.0 2.5 8.8 4.0 8.6 6.0 8.4 9.0 8.2 12.7 8.0 17.5 7.8 22.5 7.6 27.5 7.4 32.5 7.2 39.0 7.0 43.0 6.8 45.0 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). Preparation Solution Tris, 1 M stock Tris base DI Dissolve and adjust pH with 121.1 g 800 ml 70 ml 60 the following approximate amount of ml 42 ml HCl: pH 7.4 pH 7.6 pH 8.0

EDTA, 0.5 M

SSC, 20x

Disodium ethylene diamine tetraacetate 186.1 g Adjust pH to approx. 8.0 and stir until dissolved NaCl NaCitrate DI Adjust pH to 7.0 with 175.3 g 88.2 NaOH then add DI to 1 liter g 800 ml NaCl NaH2PO4 H2O EDTA DI Adjust 174 g 27.6 g 7.4 g 800 ml pH to 7.4 with NaOH then add DI to 1 liter

SSPE, 20x

TE STE (TNE)

Tris EDTA Adjust pH using Tris stock solution Tris NaCl EDTA Adjust pH to 8.0 using Tris stock solution

10 mM 1 mM 10 mM 100 mM 1 mM

GLYCINE NAOH BUFFER; PH 8.610.6, 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 8.6 2.0 8.8 3.0 9.0 4.4 9.2 6.0 9.4 8.4 9.6 11.2 9.8 13.6 10.4 19.3 10.6 22.75