Sample preparation protein denaturation

The last sample preparation method for IMS is the transfer of a tissue section onto the PVDF membrane. Proteins in the section can be transferred onto the PVDF membrane and then analyzed on the membrane. The advantage of this method is that the enzyme can be digested for MS" measurement, because the information on protein localization in the organization is fixed on the membrane.5,20 This technique can denature, reduce, and digest the proteins in the tissue section efficiently and remove the salt from the tissue. This increases the efficiency with which biological molecules are ionized, making it possible to obtain sensitive mass imaging spectra. 

Sample preparation. All allantoic fluid of chicken embryos or calf serum used in experiments contained influenza virus (104—106 EID50/ml). The samples of biological fluids underwent photodynamic treatment as described above. One milliliter aliquots were taken before treatment and at 3 and 6 h after the start of experiment. To analyze the effect of photodynamic treatment on proteins we used alkaline denaturing electrophoresis in the presence of sodium dodecylsulfate (SDS) and P-mercaptoethanol (P-ME). 

Selected entries from Methods in Enzymology [vol, page(s)] Association constant determination, 259, 444-445 buoyant mass determination, 259, 432-433, 438, 441, 443, 444 cell handling, 259, 436-437 centerpiece selection, 259, 433-434, 436 centrifuge operation, 259, 437-438 concentration distribution, 259, 431 equilibration time, estimation, 259, 438-439 molecular weight calculation, 259, 431-432, 444 nonlinear least-squares analysis of primary data, 259, 449-451 oligomerization state of proteins [determination, 259, 439-441, 443 heterogeneous association, 259, 447-448 reversibility of association, 259, 445-447] optical systems, 259, 434-435 protein denaturants, 259, 439-440 retroviral protease, analysis, 241, 123-124 sample preparation, 259, 435-436 second virial coefficient [determination, 259, 443, 448-449 nonideality contribution, 259, 448-449] sensitivity, 259, 427 stoichiometry of reaction, determination, 259, 444-445 terms and symbols, 259, 429-431 thermodynamic parameter determination, 259, 427, 443-444, 449-451. 

Effect of Sample Preparation It is well known that the viscosities of these concentrated slurries (8 percent) are shear rate dependent (14). What is less known is that the viscosity of denatured proteins are highly dependent upon dispersion conditions (Table II). For 12 percent slurries of Supro 620, viscosities decrease drastically from 10 to 33 cp as total shear is increased (1 ). If the slurries are allowed to stand, the viscosities increase slowly and revert to a much higher value (Table III). Thus, the highly sheared slurries are not at equilibrium. But, since the approach to equilibrium is slow, it is possible to use shear to produce a functionally desirable viscosity. 

The protocol involves a classical SDS-PAGE (10% polyacrylamide) run, followed by transfer onto a Western blot membrane and immunodetection with an anti-pIII antibody. Nevertheless, special care must be taken during sample preparation, because phages are very stable and difficult to denature. The protocol is similar to typical SDS-PAGE sample preparation, except that / -mer cap toe thanol should be replaced by fresh dithiothreitol (DTT, 5 mM final concentration), and the samples should be boiled in a water bath for at least 15 min. Moreover, because the pIII-fusion protein is a minor component of the virion, a large amount of phages should be loaded onto the gel, typically around 1012 phages per lane. 

Prepare the standard and unknown protein samples for analysis by mixing 10 p of each with 5 pi of 4X sample buffer in microcentrifuge tubes, mixing gently, and heating at 100°C for 5 min in a boiling water bath. This step is to ensure that all of the proteins in the sample are completely denatured. Remove the samples from the water bath and allow them to cool to room temperature. 

In these proteomics methods, the separation process is split in two phases (e.g. in Bell et al. 2001). The first phase is a protein separation by denaturing zone electrophoresis, that is in the presence of denaturing detergents, most often sodium dodecyl sulfate (SDS). The second phase is carried out by chromatography on the peptides produced by digestion of the separated proteins. This has no impact on the sample preparation itself, which just needs to be compatible with the initial zone electrophoresis. 

Prepare the denatured protein sample for loading onto the gel by adding 1 vol of sample preparation solution to 1 vol of sample at a protein concn. of approx 3 mg/mL, giving a final protein concn. of 15 mg/mL in the denatured sample. For very dilute samples (<0.25 mg/mL), use 1 vol of 5X sample preparation solution to 4 vol of sample. Boil for 5 min at lOO C, cool, and load into the sample wells on the gel. The amount of protein will vary according to source, i.e., approx 5-50 ig/track for whole cell extracts or subcellular fiactions, 0.1-1 ig/track for purified proteins (see Note 3). 

The practical solution to the protein stability dilemma is to remove the water. Lyophilization (freeze-drying) is most commonly used to prepare dehydrated proteins, which, theoretically, should have the desired long-term stability at ambient temperatures. However, as will be described in this review, recent infrared spectroscopic studies have documented that the acute freezing and dehydration stresses of lyophilization can induce protein unfolding [8-11]. Unfolding not only can lead to irreversible protein denaturation, even if the sample is rehydrated immediately, but can also reduce storage stability in the dried solid [12,13]. 

In the O Farrell method, SDS is used in the second dimension, -mercaptoethanol in the first. Others have used SDS in both dimensions and in sample preparation. The use of p-mercaptoethanol and SDS denatures proteins to polypeptides by reducing disulfide bonds and depolymeriz-ing proteins. When native proteins, such as enzymes, are desired for further study, nondenaturing sample preparation and electrophoresis conditions must be used. 

When cells are lysed, proteases (enzymes that break peptide bonds in proteins) are often activated. Degradation of proteins through protease action greatly complicates the analysis by 2D electrophoresis, so action should be taken to avoid this problem. If possible, it is advisable to inhibit proteases by disrupting the sample directly into strong denaturants such as 8 M urea, 10% trichloroacetic acid (TCA), or 2% SDS [45 7]. Proteases are less active at lower temperatures, so sample preparation at low temperature is recommended. In addition, proteolysis can often be inhibited by preparing the sample in the presence of Tris base, sodium carbonate or basic carrier ampholyte mixtures [48, 49]. 

Phospholamban was initially detected in smooth muscle in purified ER preparations from pig stomach and rabbit aorta as electrophoretic bands comigrating with the 25-kDa pentameric and 5-kDa monomeric forms of cardiac phospholamban. The dissociation of the pentamer depends on the conditions of pretreatment of the sample with the denaturing electrophoresis buffer. Phospholamban protein has also been detected in many other vascular and gastrointestinal smooth muscles, including rat aortic cells (Sarcevic et al, 1989 Karczewski et ai, 1992), canine ileum and iliac artery (Ferguson et al., 1988), and bovine aorta (L. Raeymaekers, unpublished observations Watras,... 

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