Proteins elution

Elution Proteins 0.3 M NaCl in 0.1 M (0.05 M for SWxl columns) phosphate buffer, pH 7 dextrans and PEOs distilled water. 

Dialyze eluted proteins twice in 1 liter Dialysis Buffer using Slide-A-Lyzer dialysis cassettes (Pierce) with a 30,000 molecular weight cutoff (MWTco). Because the eIF2Becat domain is 25.9 KDa, dialysis of small domains requires a lower MWTco membrane. Therefore, 15,000 MWTco Float-A-Lyzer dialysis membranes (Spectrum) are used. 

Buffer additives can overcome some of the protein interactions with the capillary wall. Some of these additives are widely used in HPLC to elute proteins off the chromatographic supports. Precautions should be taken when selecting buffer additives, especially in the areas of detector interference and their impact in the conductivity of the buffer. A list of additives used for various CZE applications can be found in Table 9.1. 

The eluted protein is taken off by a syringe from the space between sintered glass and dialysis membrane. 

If the eluted protein is used for amino acid analysis, the sample has to dialyze three times 1 h each against 100-fold of Soln. B. After lyophilization, the protein is analyzed. SDS, which is not dialyzable under the given conditions, does not influence the determination. 

Another method used for eluting proteins from an ODS column is via the salting out effect, where mobile phase gradient is run from high to low salt concentration again the most lipophilic proteins elute last. 

Despite their distinct advantages, on-line SPE and column-switching proce-dures do not always represent ideal separation techniques. In many cases, only a small number of samples can be analyzed before contamination of the precolumn by proteins occurs. Alternative techniques that prevent the adsorption of macromolecules onto column packings and allow direct injection of sample extracts are those based on use of specific LC columns. Shielded hydrophobic phase (27), small pore reversed-phase (28), and internal surface reversed-phase (29, 30) columns can be used to elute proteins in the excluded volumes, allowing small... 

Often SDS can be omitted from the elution buffer. The eluted proteins can be injected then without necessity of Triton X-100 removal. However, depending on the batch and supplier of the membrane, elution efficiency with Triton X-100 alone may vary, and some proteins are eluted poorly with this eluent. 

Brash has also used CD to study eluted proteins and finds large changes in a-helix content of fibrinogen, perhaps due to enzymatic fragmentation produced by the surface-induced activation of plasminogen to plasmin11 18). 

In some cases, stained blots are used only to identify protein band patterns while leaving the gel unmodified for subsequent steps (UNITB3.3). If such minimal protein transfer is desired, contact blotting is a suitable alternative. This unit also describes procedures for eluting proteins from membranes using detergents (Basic Protocol 2) or acidic extraction with organic solvents (Alternate Protocol 4). 

Binding affinities of most proteins to PVDF membranes are relatively strong. The most efficient protocol for protein recovery from PVDF membranes requires the use of detergents, which limits the possible use of extracted samples because detergents are often incompatible with subsequent procedures. This protocol is a simple procedure to elute proteins from the membrane into a Triton/SDS solution. A protocol that employs acidic extraction with organic solvents is also described (Alternate Protocol 4). 

Temperatures >20°C during the transfer can increase the interaction between protein and matrix and make extraction more difficult. If problems arise in eluting proteins from the membrane, try running the transfer in a cold room overnight at low currents (50 mA). 

Fit a 96-well microtiter plate (200 pL) or 2-mL collection microtubes into the vacuum manifold, carefully aligning the drop-formers to the receiver plate wells. Add 100 pL elution buffer and incubate for 2 min. Then apply a vacuum of 200-400 mbar and release vacuum after material has flowed through. Store the plate of eluted proteins at 4°C for short term or until evaluated, and then at -80°C for longterm storage. 

The samples are prepared by mixing 2 pL of eluted protein, 2 pL H20, and 2 pL of an SDS-based denaturing sample buffer containing [3-mercaptoethanol as well as upper and lower protein mass standards. Samples are heated to 95°C for 2 min and spun briefly (see Note 7). 

Separate the labeled antibody from the free dye-labeled protein using a PD-10 desalting column (Amersham Biosciences), eluting protein in 2 mL PBS to give a final antibody concentration of 0.5 mg/mL. 

Apply the sample to a micro-Mono S HPLC column and elute proteins with a salt gradient. Turn the UVdetector on 215 nm. Separate peaks manually according to absorbance at 215 nm. 

Apply fractions chosen for further purification onto a microreversed-phase HPLC column (C2/C18) and elute proteins with increasing concentrations of acetonitrile. Turn the UV-detector on 215 nm and separate peaks manually. 

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