buffers 2.pdf

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Buffers

FromRuzin, 1999. Plant Microtechnique and Microscopy

The following are recipes for a number of common biological buffers taken fromRuzin, 1999 PlantMicrotechnique and Microscopy. When choosing one for aparticular application select a buffer

based on its pH optimum and biological properties rather than its historical use. Many buffer specieshave an impact on biological systems, enzyme activities, substrates, or cofactors (Perrin andDempsey, 1974). For example, phosphate buffers inhibit the activity of several metabolic enzymesincluding carboxylase, fumarase, and phosphoglucomutase. Barbiturate uncouples oxidativephosphorylation. Tris buffer reacts with primary amines and modifies electron transport andphosphorylation in chloroplasts. Tris also inhibits respiratory enzymes in mitochondria. HEPES doesnot have these negative effects yet buffers at a similar pH range. MOPS and MES decompose whenautoclaved in the presence of glucose. Keep buffer concentration as low as possible yet enough tomaintain pH.

GLYCINEHCL; PH 2.23.6, PKA = 2.35

Combine 25 ml 0.2 M glycine andx ml HCl and dilute to 100 ml with DI (Dawson, et al., 1969).

SODIUM ACETATE; PH 3.65.6, PKA

= 4.76

Combine the following proportions of 0.1Nacetic acid and 0.1Nsodium acetate (Pearse, 1980).

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).

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

acetic acid sodiumacetate

pH

185 15 3.6

176 24 3.8

164 36 4.0

147 53 4.2126 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

Page 1 of 6Buffer tables

01/06/2011http://microscopy.berkeley.edu/Resources/instruction/buffers.html


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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 forelectron microscopy applications by Sabatini et al. (1962) as a method of avoiding adding additionalphosphates to sample preparations. Mitochondria and other organelles can be damaged whenexposed to the high concentrations of phosphates present in Srensens buffers. Also, cacodylate willnot react with aldehyde fixatives as will amine-containing buffers (e.g., Tris). Its efficacy in fixationsolutions may be a result of the metabolism-inhibiting effect of the arsenate rather than any specialbuffering capacity.Prepare a 0.2 M stock solution of sodium cacodylate in water (4.28 g/100 ml). Add the followingamounts of 0.2 M HCl per 100 ml cacodylate stock solution, followed by the addition of DI to a finalvolume of 400 ml, to obtain 0.05 M cacodylate buffer at the desired pH (Dawes, 1971).

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 beused successfully to buffer staining solutions (e.g., Toluidine Blue O). Avoid Tris with aldehydefixatives 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 forretention of actin filaments during fixation. Other useful biological buffers include HEPES (pKa =

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

(7H2O) DI

43.2 g 17.5 mM

to 1 liter

Use TBS when performing

immunocytochemicalexperiments 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)

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 6.618.6 6.8

12.6 7.0

8.4 7.2

5.4 7.4




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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. Usex ml A +y ml B and dilute to 100 ml with 50 ml DI.

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 MSrensens phosphate buffer solution (Srensen, 1909; Gomori, 1955). Keep in mind that high levelsof phosphate may be somewhat toxic to plant cells (Sabatini, et al., 1962) and thus Srensens buffermay not be appropriate for some experiments.Stock solutions: A 0.2 M NaH2PO4 B 0.2 M Na2HPO4

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 4.623.0 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

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




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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 are0.2 M dibasic sodium phosphate; 0.1 M citric acid (Pearse, 1980).

BARBITAL BUFFER; PH 6.89.2, 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) addx ml 0.2 M HCl to create the buffered solution and dilute to 200 ml with DI(Gomori, 1955).

16.0 84.0 7.5

8.5 91.5 7.8

5.3 94.7 8.0

0.2 MNa2HPO4(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 3.416.1 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

0.2 M HCl (ml) pH

1.5 9.2

2.5 9.0

4.0 8.8

6.0 8.6




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TRIS BUFFERS

Tris buffers are used commonly in microtechnique applications involving molecular biologicalprocedures. Listed here are a number of common Tris formulations (Maniatis, et al., 1982).

GLYCINE NAOH BUFFER; PH 8.610.6, PKA = 9.78

Stock solutions:0.2 M glycine0.2 NaOHCombine 25 ml glycine stock solution withx ml 0.2 M NaOH and dilute with DI to make a 100 mlsolution (Pearse, 1980).

9.0 8.4

12.7 8.2

17.5 8.0

22.5 7.8

27.5 7.6

32.5 7.439.0 7.2

43.0 7.0

45.0 6.8

Solution Preparation

Tris, 1 M stock Tris base DI Dissolve and adjust pH withthe following approximate amount ofHCl: pH 7.4 pH 7.6 pH 8.0

121.1 g 800ml 70 ml 60ml 42 ml

EDTA, 0.5 M Disodium ethylene diamine tetraacetateAdjust pH to approx. 8.0 and stir untildissolved

186.1 g

SSC, 20x NaCl NaCitrate DI Adjust pH to 7.0 withNaOH then add DI to 1 liter

175.3 g 88.2g 800 ml

SSPE, 20x NaCl NaH2PO4 H2O EDTA DI AdjustpH to 7.4 with NaOH then add DI to 1liter

174 g 27.6 g7.4 g 800ml

TE Tris EDTA Adjust pH using Tris stocksolution

10 mM 1mM

STE (TNE) Tris NaCl EDTA Adjust pH to 8.0 usingTris stock solution

10 mM 100mM 1 mM

0.2 M NaOH pH

2.0 8.6

3.0 8.8




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4.4 9.0

6.0 9.2

8.4 9.4

11.2 9.6

13.6 9.8

19.3 10.422.75 10.6


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