Barbital buffer

The presence of Individual chains In a hemoglobin variant can also be demonstrated by electrophoresis at alkaline pH after the protein has been dissociated Into Its subunits through exposure to 6 M urea In the presence of 3-mercaptoethanol. The buffer is either a barbital buffer or a tris-EDTA-boric acid buffer, pH 8.0 - 8.6, and contains 6 M urea and 3-niercapto-ethanol. Dissociation of the hemoglobin Into subunits Is best accomplished In a mixture of 1 ml 10 g% Hb (or whole hemolysate), 4 ml 6 M urea barbital or tris-EDTA-boric acid buffer, and 1 to 1.5 ml 3-mercaptoethanol. After 30 minutes to 1 hour the sample Is subjected to cellulose acetate or starch gel electrophoresis. Each chain has a specific mobility and an alteration In electrophoretic mobility easily Identifies the abnormal chain. 

M barbital buffer, pH 8.6 cellulose acetate 200v for 2 hours Ponceau S. 182... 

Aliquots (2-5 pi) of serum are applied to an agarose gel using a sample application template. Incubation for 5-10 min allows diffusion of the sample into the gel before the gel is transferred to the electrophoresis unit. Depending on the gel size and commercial application, electrophoresis is performed at 50-80 V for 45-90 minutes in barbital buffer. The gel is then dried in an oven at 60-80°C for 10-20 min. 

In the first dimension, 20 [tl plasma is separated by electrophoresis at 4°C in a 0.75% agarose gel using a 1 2 16 dilution of a barbital buffer. Bromophenol blue is added to a standard sample to visualize albumin in the native gel. The electrophoresis is stopped when the albumin/bromophenol blue marker has migrated 6 cm. Agarose gel strips containing the preseparated lipoproteins are then transferred to a 4-20% polyacrylamide gradient gel. Separation in the second dimension is performed at 40 mA for... 

Several compositions for buffer with a pH about 8.6 have been published (1), most of them containing barbital. The most commonly used is barbital buffer. 

Migration of plasma lipoproteins on paper electrophoresis at pH 8.6 in barbital buffer. LP = Lipoprotein other abbreviations as in Table 20-1. 

Titan Gel lEP plate No. 3047. Plates contain 1% agarose (w/v) in barbital-sodium barbital buffer with 0.1% sodium azide added as a preservative. The plates are ready for use as packaged and should be stored horizontally at 15 to 30 C. Discard plates if they appear cloudy, show bacterial growth, or have been frozen or in excessive heat. 

The many hydroxyl groups on cellulose provide for extensive interaction between polar macromolecules and the paper electrophoresis strip. Therefore, hydrophilic proteins and nucleic acids tend to have low electrophoretic mobility. Acetylation of the cellulose hydroxyl groups produces a medium (cellulose acetate) that greatly speeds up electrophoreses. For cellulose acetate electrophoreses O.IM barbital buffer is the standard one. 

S M barbital-sodium barbital buffer (pH 8.6) (for plasma proteins) containing 3 mM calcium lactate. The gels are prepared by suspending 1.5 g of agarose in 100 ml of barbital buffer and the suspension is heated until a clean solution is obtained. Then the agarose solution is cooled to 60°C, and 25 ml are poured over a... 

The optimum pH of rat serum lipase lies between 7.2 and 8.05, depending on the substrate (26). For rabbit serum and hiunan serum lipases the optimum pH depends on the bufFer (271). Thus the optimum pH range of rabbit serum lipase is 8.6-9.8 in glycine buffer but 7.4-8.0 in barbital acetate buffer, while for human serum lipase the pH optimum in glycine buffer is more alkaline than 13, in barbital buffer 7.7-8.0, and in phosphate buffer 9.2 (271). 

FIGURE 2. Plot of CL intensity versus time. Each vial contained PMN, luminol, and autologous serum suspended in barbital buffer, pH 7.2, to a final volume of 2.0 ml. Zymosan was added to each vial 30 seconds before the fourth cycle count. Curves a through d represent the CL responses from 10,000, 20,000, 30,000 and 40,000 PMN, respectively. 

FIGURE 8. Plot of integral CL versus time. Each curve depicts the luminol-amplified CL response from a suspension of human PMN in barbital buffer, pH 7.2, following addition of P. aeruginosa. Curve a did not receive serum curves b through e received the quantities of pre-imaune and immune rabbit serum indicated. 

By paper electrophoresis in pH-8.6 barbital buffer of ionic strength 0.05—0.06. -41, 81, 83, 89. 95, 111 Hemoglobin H is the fastest and Cthe slowest. The numbers have no quantitative significance. 

Fig. 2. Reaction of 3 -p-fluorosulfonylbenzoyladenosine with bovine liver glutamate dehydrogenase. Glutamate dehydrogenase (021 mg/ml) was incubated with 3 -FSBA (0.496 mil/) at 24° in 0.01 M sodium barbital buffer (pH 8) containing 0.43 M KCl and 5% ethanol. At each indicated time, an aliquot was withdrawn, diluted 20-fold with Tris-0.1 M acetate buffer (pH 8) at 0°, and assayed (A) in the absence and (B) in the presence of 100 yM ADP. Inset Determination of the pseudo first-order rate constant from the decrease in activation by ADP. (Ft and Fo are the enzymic velocities measured in the presence of ADP and the given and zero time, respectively, and F > is the constant velocity at the end of the reaction. The pseudo first-order rate constant calculated is 0D351 min. ) Data are taken from P. K. Pal, W. J. Wechter, and R. F. Colman, Biochemistry 14, 707 (1975).

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