TRICINE buffer

Hall, M. S., and Leach, F. R. (1988). Stability of firefly luciferase in Tricine buffer and in a commercial enzyme stabilizer. /. Biolumin. Chemilumin. 2 41-44. 

Chloroplasts will be isolated by careful extraction of spinach leaves, using tricine buffer containing sucrose. The crude extract contains both whole and fragmented chloroplasts, but both contain all the necessary photosyn-thetic components and are capable of photophosphorylation. The preparation described in this experiment retains almost all of the chlorophyll in the chloroplasts. The total chlorophyll content of the chloroplasts will be determined by extracting the pigment with aqueous acetone and measuring the absorption at A. = 652 nm. The chlorophyll concentration is calculated according to Equation E9.3 (Arnon, 1949),... 

Alternate Protocol 3 SDS-PAGE in a Tris-Tricine Buffer System B3.1.9... 

The Tris-glycine discontinuous buffer system of Laemmli cannot be used for the separation of proteins with molecular weights < 10 to 15 kDa. For the analysis of smaller proteins, an alternative Tris-tricine buffer system is used along with an acrylamide solution that has a high percentage of cross-linker. This technique should be used when separating peptides in the size range of 1 to 20 kDa. 

Tricine buffer (114 mM) dissolve 2 g AH lris(hydroxymcthyl)methyl Iglycinc (Tricine, from Sigma) in 100 mL Milli-Q deoxygenated water (see Note 2). 

Add 5 mCi 99mTc04 to a glass 10-mL tube with 0.3 mL Tricine buffer, cover with parafilm, and purge with nitrogen for 15 min. 

Check out pH of Tricine buffer, it should be 6.0 2. If necessary, adjust pH with small amounts of 1MNaOH or 1MHC1 until pH reaches 6.0 2. For making this solution,... 

Figure 18 Effect of nonionic detergent (Triton X-100) on benzydamine N-oxygenation (FMO) and N-demethylation (CYP) by human liver microsomes. Benzydamine (500 pM) was incubated with pooled human liver microsomes (1.0 mg protein/mL) in tricine buffer (50 mM, pH 8.5 at 37°C) with or without Triton X-100 [1% (v/v)]. Reactions were initiated by the addition of an NADPH-generating system and stopped after 10 minute by the addition of an equal volume (500 pL) of methanol. Precipitated protein was removed by centrifugation, and an aliquot (25 pL) of the supernatant fraction was analyzed by HPLC with fluorescence detection. Abbreviations FMO, flavin monooxygenase CYP, cytochrome P450.

Figure 19. Resonance Raman spectra of the oxidized Mo domain of recombinant human SO. (a) The Mo domain prepared by tryptic cleavage of the K108R variant of the holoenzyme. (b) His-tagged Mo-domain. Spectra recorded using 488-nm excitation for samples (0.5-1.0 mM in Mo) in 50-mM tricine buffer, pH 8.0, frozen at 17-25 K. Bands marked an asterisk correspond to lattice modes of ice and bands marked with P correspond to nonresonantly enhanced protein modes. A linear ramp has been subtracted to correct for a sloping fluorescence background.

Prepare and chill 500 ml grinding medium consisting of the following components 0.4M sucrose, 0.165M Tricine buffer adjusted to pH 7.5, O.OIM KCl, O.OIM MgCU, O.OIM EDTA adjusted to pH 7.5, and O.OIM dithiothreitol. 

The solution as mobile phase (imidazole lOOmmol/L in the Tricine buffer 50 mmol/L, pH 9.4) was flowed at 0.1 mL/min using HPLC pump to the flow cell reactor via inlet connecting tube, and drained via a outlet tube. H2O2 specimen (20 juL) was injected with a loop injector (7125, JASCO). The reaction temperature for CL was room temperature. Light from the flow-through chip was detected after the dark frame reduction with the CCD-CL monitor. 

Determination of pyrogallol. Pyrogallol was assayed by HRP catalyzed imidazole chemiluminescence coupled to the micro-flow injection system at room temperature. Pyrogallol specimens (50 pL) were injected using an autosampler (AS-950, JASCO, Tokyo, Japan) every five min into a stream of water (100 uL/min) using a HPLC pump (PU-980, JASCO), and the other mobile phase (imidazole 100 mmol/L in the Tricine buffer 50 mmol/L, pH 9.3) was delivered at 100 pL/min. The light emitted from the reactor tube was detected with a... 

Figure 11. Electropherogram of protein standards run in an untreated fused silica capillary, in pH 8.24 tricine buffer.

The coupling of the CZE step to detection systems other than UV has required the development of separation conditions compatible to the detection system used. For instance, the presence of primary amines, such as DAB, in buffers needed to be avoided for compatibility with laser-induced fluorescence (LIE) of compounds derivatized with fluorogenic substrates through their amino groups [90]. Baseline resolution of eight peaks in approximately the same time was achieved by substituting DAB by morpholine and tricine by boric acid (to avoid potential traces of primary amines in the tricine buffer) and by adjusting the concentration of other buffer components to compensate for the increase in electrical current. In the same work, modifications were also required to achieve compatibility with MS detection where nonvolatile salts, urea, and amines should be usually avoided. A physically adsorbed polyethylenimine-coated capillary was used to overcome protein adsorption to the capillary walls in the absence of cationic additives and the use of an acetate buffer at pH 5.05 allowed the partial resolution of at least five bands of rhEPO. Other types of coated capillaries have been used for the analysis of EPO by CE-MS as detailed in Section 22.4.3.3 [30,37,42,62,96]. 

Preparation of crude cell-free extracts Synechococcus PCC 6301 was grown photo-autotrophically (14) and cells were broken using a French press at 86.25 MPa. in 0.1 M Tricine buffer pH 9.0, containing 0.3 M glycerol, 25 mM MgCl2 and 1 mM dithiothreitol (DTP). After centrifugation at 200,(XX) g (20 min) the supernatant was used for further purification. 

To evaluate the photosystem 2 (PS2) activity the parameters Fg and Fy of prompt fluorescence induction kinetics of leaf discs and isolated chloroplasts were measured with chlorophyll fluorescence measuring system, PAM (15). The chloroplasts were diluted with 10 mM Tricine buffer, pH 7.8, containing 330 mM sorbitol, 5 mM MgSOd and 1.6 mM of corresponding PA to 60 pg/ml. The leaf discs were incubated 1h in... 

P(LLA-co-3-methyl- 37 Tris, tricine-buffered solution Solid [179]... 

Koh et al. used tricine buffer, pH 8.8, uncoated capillary 37 cm X 75 pm, and 11 kV voltage for separating ascorbic acid and lAA (internal standard) from urine, plasma, and serum (60). The capillary was treated with NaOH before use. They used either MPA or TCA for stabilizing ascorbic acid in the sample before the CE run. However, as found later, TCA may result in the loss of nearly 40% of the ascorbic acid content (63). The between-day coefficient of variation was 1.9% to 3.3%. The detection limit was 1.6 pg/mL and the method was linear to 480 pg/mL (60). 

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