Buffer combinations

Some cell lines, such as HEK293, may detach during the permeabilization step due to a strong Ca2+ dependence for attachment. While it is critical that cells are permeabilized as a monolayer, detachment does not seem to hinder cytosolic ribosome release, as they tend to detach as a (partial) monolayer. Following the permeabilization step, cells can simply be separated from the soluble cytosol phase by centrifugation at 750-1000 Xg for 5 min. Transfer the supernatant (cytosol) to a new tube. Remove any remaining cells attached to the flask via the wash buffer, combine with the cell pellet, and recover by centrifugation. Proceed with the membrane solubilization step. 

The sodium acetate-acetic acid combination is one of the most widely used buffers, and is usually referred to simply as acetate buffer. Other buffer combinations commonly employed in chemistry and biochemistry include carbonate-bicarbonate (sodium carbonate-sodium hydrogen carbonate), citrate (citric acid-trisodium citrate), phosphate (sodium dihydrogen phosphate-disodium hydrogen phosphate), and tris [tris(hydroxymethyl)amino-methane-HCl]. 

Centrifuge at low speed (e.g., 1000 x g) 10 min at room temperature to recover the cell walls. Resuspend the pellet in 50 ml of 20 mM HEPES buffer, pH 6.7. Wash twice more in buffer. Combine with all the DMSO supernatants and dialyze the supernatants. 

Optional Resuspend the pellet in 50 ml 20 mM HEPES buffer, pH 6.9/2 mM CaCl2. Incubate with 200 U porcine pancreatic a-amylase at 40°C for 1 hr with stirring. Filter on 1 l-p,m mesh in a Buchner funnel. Wash twice with 50 ml HEPES/CaCl2 buffer, combine the filtrates. 

Filter the cell walls on 1 l- J,m nylon mesh. Wash twice with 20 ml of 20 mM HEPES buffer, combining the a-amylase treatment filtrates. 

Acetate Buffer Combine 50 mL of 2.0 N Acetic Acid and 11.3 mL of 4.0 N Sodium Hydroxide in a 1000-mL volumetric flask, and dilute to volume with water. Verify that the pH is 4.50 0.05, using a pH meter, and adjust, if necessary, with... 

Buffers—Combinations of acids, bases, and salts that neutralize changes in pH. 

Fig. 13 The dissolution behavior of triamterene (TRIAM) (A) and hydrochlorothiazide (HCTZ) (B) formulations containing glycine and citric acid (Formula D) on storage at 40 C/85% RH up to 12 weeks. This figure is correlated to Fig. 7 and is from the same reference. The overlapping curves in both cases show that the amino acid/buffer combination is able to neutralize the adverse effect of storage under high humidity and temperature. The figure further depicts that neutralization remains effective even on storing the capsules as long as 12 weeks. (From Ref. p. 498 by courtesy of Marcel Dekker, Inc.)...

The sample resistance, R, depends on the exact buffer and salt conditions used to solubilize the peptide or protein. Although as a general rule the value of Rs will increase, and the SNR will correspondingly decrease, as the sample ionic strength is increased, R is more strictly related to the sample conductivity (ionic strength, the one with lower conductivity will yield the best SNR. In theory, then, dissolving the protein of interest in H20 would provide the best SNR. However, for proteins and peptides, it is often impractical to use H20 or a salt/buffer combination with very low conductivity since many proteins are salted in at moderate salt concentrations (see Chapter 9.12). Thus, the choice of buffer will... 

Blottinpf buffer. Combine blocking buffer (1 part) with wash buffer (3 parts) just prior to use. 

Dye Buffer Combine 2.5 mL of sample buffer, 2.0 mL of glycerol, 400 pL of (3-mercaptoethanol, and 200 pL of a saturated solution of bromophenol blue. This buffer should be made fresh for each mn. A stock solution can be made without adding the (3-mercaptoethanol. This should be added just prior to use. 

The following conditions work well with Hybond N and N (Amersham) membranes using a prehybridization/hybridization buffer made according to the following recipe. For 50 ml of buffer, combine 40 ml sterile distilled... 

Buffer solution Solution of an acid plus a base that controls pH by reacting with added base or acid Buffer Combination of an acid and a base that can control pH... 

The sheathless design has the advantages of high detection sensitivity, low flow rates, long-term stability, and the absence of interference from the sheath liquids. A major concern of this design is to find an optimum buffer combination for CE separation and ESI operation. 

The RFID-based model to overcome the aforementioned deficiencies involves implementing a facility-wide decision-making model that utilizes RFID data coming from tagged inventory items stored in the buffers. The location of each buffer combined with time-stamped tag ID can be used to capture the actual status of each buffer and incoming and outgoing materials, thus inventory replenishment can be carried out in a more efficient manner. 

A buffer solution has a built-in mechanism that counteracts the effect of adding acid or base. Consider the effect of adding HCl or NaOH to an acetic acid-sodium acetate buffer. When a small amount of HCl is added, the acetate ions of the buffer combine with the H ions from HCl to form un-ionized acetic acid, thus neutralizing... 

Desacetylcephapirin was extracted from milk samples and quantitated using a PLRP-S column (A = 290 nm) using a 16/84 acetonitrile/water (10 mM H3PO4 with lOmM KH2PO4 and lOmM sodium decanesulfonate) mobile phase [1379], This study also tabulated the conditions for the analysis of amoxicillin, ampicillin, cephapirin, ceftiofur, cloxacillin, and penicillins G and V. Again a C,g and an 30% acetonitrile/water (phosphate buffer) combination were used in each case. 

An important component of blood is the buffer combination of dihydrogen phosphate ion and the hydrogen phosphate ion. Consider blood with a pH of 7.44. 

SPE benzodiazepine wash solvent containing 20% acetonitrile/0.1 M phosphate buffer Combine 80 mL of... 

Capillaiy zone electrophoresis (c.z.e.) separation of anions including carboxylates of sugar acids was achieved in a 3 minute run with indirect u.v. detection using a complex co-ion as an electroosmotic flow modifter that dynamically coats the capillary walls. " Inositol mono-, bis-, tris- and hexakis-phosphates were similarly separated using a tetradecylammonium bromide - borate buffer combination. Inositol 1- and 2-phosphate were easily separated." ... 

The software has been equipped with a fimction such that it is not only able to conduct experiments with one column/organic modifier/buffer combination, but to automatically optimize a method by trying different column/organic modifier/ buffer combinations. The system provides imattended HPLC method development and performs autonomous development and optimization of isocratic and gradient methods for selection of the best variant column, pH value, solvent Two typical hardware configurations and other mixed combinations are supported by ChromSword standard and powerful. 

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