Washing buffers

The pectinase activity was found in void volume of the washing buffer while some contaminated protein was adsorbed to the ion exchanger (Figure 1. The activity was 100% recovered. Thoug i, the enzyme was not adsorbed to the DEAE-ceUulose, but it is useful since the some contaminated proteins coidd be separated. The enzyme was approx. 10 fold purified and its specific activity increased to 48.8 unit/mg protein (Table 1). 

Elution buffer identical to wash buffer, but additionally containing 3 xFLAG peptide (Sigma) to a final concentration of 0.1 mg/ml... 

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. 

Quickly transfer the slide (without removing the coverslip) into a rack and submerge it in Wash buffer 1 (2 X SSC and 0.05% SDS). Similarly, transfer each of the other slides into the soaked rack. Use needle to gently assist with the falling of the coverslips from the hybridization areas of the slides. Be careful not to touch any of the printed area. Shake the slides for 5 min at room temperature. 

Transfer the rack into Wash buffer 2 (1 x SSC). Absorb excess Wash buffer 1 with a paper towel before putting the rack in Wash buffer 2. Shake the slides for 5 min at room temperature. 

Prepare a chitin affinity column by washing with at least 10 bed volumes of 25 mM HEPES, 250mM NaCl, ImM EDTA, 0.1 percent Triton X-100, pH 7.0 (wash buffer). 

Recover the cells by centrifugation, lyse them in wash buffer containing protease inhibitors (e.g., 20 pM PMSF), and clarify the supernatant by centrifugation. 

Apply the lysate onto the affinity column and wash with at least 10 column volumes of wash buffer to remove not-bound protein. Monitor the eluate by absorbance at 280 nm to assure that baseline has been reached. 

Elute the conjugated molecules using wash buffer and purify using dialysis or gel filtration. 

This procedure can be repeated for other coating conditions (time, temperature, pH, buffer compositions, blocking steps, wash buffers, etc.). 

For longer storage, air-dried cryosections are best kept at 20°C or at 80°C until needed. When required, allow the slides to warm at room temperature for 5 min, then fix again in acetone (optionally) for 5 min and rinse in a washing buffer of choice. 

The feed is introduced at the top of the annular column at the 0° position. The feed solution is followed by a wash buffer, which is introduced to the annular column through the main inlet port. A 1 vol.% mixture of 2-propanol in a 100 mmol/1 ammonium acetate buffer was used as wash buffer. In the washing zone the nicked DNA followed by the RNA are eluted from the column according to their affinity to the resin. At 180° offset from the feed nozzle the elution buffer (5 vol.%) 2-propanol in 100 mmol/1 ammonium acetate) was pumped to the annulus of the column. The elution buffer was used to strip off the bounded Plasmid DNA. Regeneration of the column was achieved by a 20 vol.% mixture of 2-propanol in 100 mmol/1 ammonium acetate buffer. All of the above-mentioned steps, i. e., feed, wash, elution, and regeneration, were done simultaneously and continuously on the P-CAC system. 

Figure 3.33 Capillary electrophoresis. The sample is introduced through the inlet, washed in with a wash buffer and then separated under the influence of a potential difference between the two electrodes. The zones are monitored as they pass through the detector and the data captured and computed.

The equilibration buffer or wash buffer is applied on the column at a monitored flow rate. The eluate is collected as the wash. For size exclusion, the eluates are collected in fractions. 

Hybridization and washing buffer Hybrisol Vll (Oncor, Gaithersburg, MD), which is a mixture of formamide (50%) in SSC (2X). 

Repeat step 2 using fresh wash buffer. 

Unfortunately, the conditions for the immunoprecipitations (especially the stringency of the binding and wash buffers) have to be determined experimentally for each antibody. These stringent conditions, on the one hand, can help to reduce the background, but on the other hand, can result in a loss of signal. 

Chromatography column 20 ml bed volume Wash buffer 50 mM sodium phosphate, 300 mM NaCI,... 

Wash with 20 ml of the wash buffer and collect 5 ml fractions checking A280nmuntil it is <0.01. 

Add 200 pel of Wash Buffer to each well, remove from the magnet, and mix on the microplate shaker (or vortex) for 5 min. 

Eguilibrate with 2 x column volume Binding/Wash Buffer (BOm M Tris pH 7.5, BOO mM NaCI, 20 mM imidazole). 

Wash the column with 5 x column volume Binding/ Wash Buffer or until the absorbance at 280 nm becomes stable. 

Wash the column with 2 x column volume of wash buffer. 

Wash the column with 10 x column volume of wash buffer to removed proteins which have bound non-specifically to the resin. 

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