BUFFERS 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 2 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, PK, = 2.35 Combine 25 mi 0.2 M glycine and x ml HCI and dilute to 100 mi with DI (Dawson, et a/., 1969). x (ml) pH 22.0 2.2 16.2 24 42.4 2.6 34 28 5.7 > 4. 3.2 3.2 34 25 3.6 SODIUM ACETATE; PH 3.6-5.6, PK, = 4.76 ‘Combine the following proportions of 0.1N acetic acid and 0.1N sodium acetate (Pearse, 1980). acetic sodium pH acid acetate 185 15 3.6 176 24 3.8 164 36 40 147 53 42 126 74 44 102 98 46 80 120 48 5.0“GOOD” BUFFEI Tris-HCl (pKa = t used successfully fixatives or osmiu BUFFERED SALINE (PBS, TBS, TNT, PBT) Buffered saline solutions are used frequently when pet ‘There are many variations. Presented here are three common ming immunolocalization experiments, crormon formulations (Mishkind, etal, 1987). PBS 20x stock te Potassium chloride 49 53.6 mM Potassium chloride 4 9 Nac 160g 274mM NaCl 1609 i Potassium phosphate monobasic 4g 29.4 mM Tris buffer (10 mM, pH 7.5) to liter ‘Sodium phosphate dibasic 43.29 17.5 mM Use TBS when performing (7*H20) DI to 1 liter 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 Lliter Tween 20 1% (v/v) Sodium cacodylate buffer [Na( is a alternative It has good pH buffering capacity within the range of pH \codylate 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 Sorensen’s buffers. Also, cacodylate will not react with aldehyde fixatives as will amine-containing buffers (e.9., Tris). Its efficacy in fix lutions may be a result of the metabolism-inhibiting effect of the arsenate rather than ai capacity. Prepare a 0. jum cacodylate in water (4.28 g/100 ml). Add the following amounts of ck solution, followed by the addition of DI to a final volume of 4 rat the desired pH (Dawes, 1971). pH 5.0 5.2 54 5.6 5.8 6.0 6.2 6.4 66 68 7.0 7.2 74“GOOD” BUFFERS; PK, = 6,15-8.06 Tris-HCl (pKa = ; y ey Dene ae maleate (pKa = 6.26) have a working range of pH 5.0-8.6 and may be uuffer staining solutions (e.g., Toluidine Blue 0). Avoid Tris with aldehyde fixatives or osmium tetroxid le, however, as the aldehyde i f Tris re a yydes reacts with the amino group of Tris, rset In the less of buffering capacity. PIPES (pKa = 6.80) Is commonly used as a buffer for 7.55), MES (pKa laments during fixation, Other useful biological buffers include HEPES (pKa = A (pKa = 6.15), and MOPS (pKa = 7.20) (Good, et al., 1966; Perrin and Dempsey, 1974), Mit Mi itrate buffer (Gomori, 1955) stock solutions: A: 0.1 ‘sodium citrate. Use x ml A + y ml B and dilute to 100 ml with 50 ml DI. 0.1M 0.1M pH citric sodium acid citrate 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 ee eee ee 28.0 22.0 44 oe ae (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 enum items 2 42.8 6.2 illed water to make a final 0.1 M Sorensen’s. phosphate buffer solution (Sorensen, 1909; Gomori, 1955). Keep in mind that high Ievels of phosphate may be somewhat toxic to plant cells (Sabatini, et a/., 1962) and thus ‘Sorensen’s buffer may not be appropriate for some experiments. Stock solutions: A 0.2 M NaH2PO4 B 0.2 M NazHPO4 Mix appropriate volum A(ml) B(mI) pH 92.0 8.0 5.8 87.7 12.3 6.0 815 18.5 6.2 68.5 31.5 6.5 62.5 37.5 6.6 56.5 43.5 6751.0 49.0 Ge 45.0 55.0 6.9 39.0 61.0 Zo 33.0 67.0 aa 28.0 72.0 7.2 23.0 77.0 73 19.0 81.0 74 16.0 84.0 75 85 91.5 78 5.3 94.7 8.0 PHOSPHATE-CITRATE BUFFER; PH 2.2-8.0, PK, = 7.20/6.40 Add the following to create 100 mi of phosphate/citrate buffer solution, Stock solutions are 0.2 M dibasic sodium phosphate; 0.1 M citric acid (Pearse, 1980). 0.2M 01M pH Na2HP04 citrate (ml) (ml) 5.4 44.6 26 7.8 42.2 2.8 20.2 39.8 3.0 12.3 37.7 3.2 144 35.9 34 16.2 33.9 3.6 17.7 32.3 38 19.3 30.7 4.0 20.6 29.4 42, 22.2 27.8 44 23.3 26.7 46 24.8 25.2 48 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 Geese 33.4 16.9 6.2 34.6 15.4 64 36.4 13.6 6.6 40.9 ot 68 43.6 65 7.0Add the following ‘BARBITAL BUFFER; PH 6.8-9.2, PK, = 98 to create 200 mi of buffered solution. To 50 ml of 0.2 M sodium barbital (Veronal, 41.29 in 1000 ml) add x ml 0.2 M HCI to create the buffered solution and dilute to 200 ml with DT (Gomori, 1955). ‘TRIS BUFFERS 0.2 M HCI (ml) LS 25 40 6.0 9.0 12.7 17.5 225 27.5 32.5 39.0 43.0 45.0 pH 9.2 9.0 8.8 8.6 8.4 8.2 8.0 78 76 74 7.2 7.0 68 Tris buffers are used commonly in microtechnique applications involving molecular biological procedures. Listed here are a number of common Tris formulations (Maniatis, et a/., 1982). Solution Tris, 1M stock Tris base DI Dissolve and adjust pH with the following approximate amount of HCl: pH 7.4 pH 7.6 pH 8.0 EDTA, 0.5 M ‘ssc, 20x SSPE, 20x t TE STE (TNE) Preparation Disodium ethylene diamine tetraacetate Adjust pH to approx. 8.0 and stir until dissolved NaCl NaCitrate DI Adjust pH to 7.0 with NaOH then add DI to 1 liter NaCl NaH2PO4 * H20 EDTA DI Adjust pH to 7.4 with NaOH then add DI to 1 liter Tris EDTA Adjust pH using Tris stock solution Tris NaCl EDTA Adjust pH to 8.0 using Tris stock solution 121.19 800ml 70m! 60 mi 42ml 186.19 175.39 88.29 800ml 174.9 27.697.49 800 mi 10 mM 1 mM 10mM 100mM 1 mM decl5@ hatmai}.com Codhdtecc QyeedheyGLYCINE- NAOH BUFFER; PH 8.6-10.6, PK, = 9.78 Stock solutions: 0.2 M glycine 0.2 NaOH’ Combine 25 mi glycine stock solution with x ml 0.2 M NaOH and dilute with DI to make a 100 ml solution (Pearse, 1980). fu 010mg fob 7 \Wha,g0, SAV mg/m b EY 0.2 M NaOH 2.0 3.0 44 6.0 8.4 11.2 13.6 19.3 22.75 pH 8.6 8.8 9.0 9.2 9.4 9.6 98 10.4 10.6 tate tate