Proteomic profiling of large myofibrillar proteins from dried and long-term stored polyacrylamide gels.

A method for the utilization of dried polyacrylamide gels from the pre-proteomic era is described in order to enable the mass spectrometric analysis of long-term stored protein preparations. The in-gel digestion of high-molecular-mass proteins embedded in a 20-year old gel was carried out following gel re-swelling and resulted in the proteomic identification of a large number of proteins, including 3400 kDa titin, 800 kDa nebulin and myosin heavy chains of 220 kDa from rabbit skeletal muscle. These findings demonstrate that dried protein gels from past biochemical analyses can be successfully reused and analyzed by modern and refined mass spectrometric techniques.

Fast reversible single-step method for enhanced band contrast of polyacrylamide gels for automated detection.

Staining SDS-PAGE is commonly used in protein analysis for many downstream characterization processes. Although staining and destaining protocols can be adjusted, they can be laborious, and faint bands often become false negatives. Similarly, these faint bands hinder automated software band detections that are necessary for quantitative analyses. To overcome these problems, we describe a single-step rapid and reversible method to increase (up to 500%) band contrast in stained gels. Through the use of alcohols, we improved band detection and facilitated gel storage by drying the gels into compact white sheets. This method is suitable for all stained SDS-PAGE gels, including gradient gels and is shown to improve automated band detection by enhanced band contrast.

A simple drying solution that minimizes cracking during air-drying of polyacrylamide gels.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis is a routine technique used in biochemistry. Air-drying is an economical method of gel preservation that does not require expensive equipment. Our laboratory uses drying frames from RPI, which recommends a drying solution of 20% ethanol and 10% glycerol. The solution performs well for gels up to 10% acrylamide and 0.75 mm thickness; however, crack formation may occur if nicks or bubbles are present. The literature shows various drying methods and combinations of alcohol (30-100%) and glycerol (5-35%), but still reports cracking problems. Tests were conducted to independently evaluate the effects of ethanol and glycerol concentration on gel cracking. Here we introduce a simple solution that does not require glycerol or modified frames to generate preserved, crack-free sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.

A simple method of drying polyacrylamide slab gels that eliminates cracking.

The polyacrylamide slab gel is the most common gel format for analyzing protein samples by electrophoresis. Drying these gels is useful in many biological applications; for example, autoradiography, in which radiolabeled proteins are separated to enable their detection and identification. Dried protein gels can also serve as an ideal method of preserving the gel itself for permanent record-keeping and allowing densitometry at a convenient time. Here I describe a simple and highly reproducible gel-drying method that results in dried gels without the cracks that are frequently encountered with many existing gel-drying methods.

Gel Drying Methods.

There are several reasons for drying of polyacrylamide gels after gel electrophoresis; for example, it is necessary if autoradiography has to be performed using radioactive labeled proteins. Another reason may be to simply store the gel in the laboratory book. Aside laborious commercial solutions, the simple and cheap drying protocol presented here may be sufficient especially for storage of the dried gel in the lab book.

Effects of desiccation on Euterpe edulis Martius seeds

Information on desiccation sensitivity of Euterpe edulis seeds under two drying rates is presented. The sensitivity was studied during the course of germination and normal germination. The water content was evaluated for both seeds and embryos. Results showed the following: (a) For both drying treatments and for both germination and normal germination, desiccation sensitivity values were higher for measurements based on the water content of the embryo than for those of the seed. (b) For both drying treatments, desiccation sensitivity were higher for normal germination than for germination based on both the embryo and seed water contents. (c) Under the slow drying treatment and for measurements based on the seed water content, critical water content was visible for normal germination but not for germination; (d) Critical water contents for germination and normal germination were more clearly established in the fast drying treatment than they were in the slow drying method based on both the embryo and seed water contents. Critical water contents were not associated with changes in electrolyte leakage, which suggests that conductivity is not a good indicator of physiological seed quality. From the beginning of both drying treatments, changes in nuclei and vacuoles were observed, but, when seed water content was reduced to below critical values, the cells became severely plasmolyzed, the vacuoles highly distorted, and the nuclei formed an almost homogeneous mass with the chromatin and the nucleoplasm, which suggests irreversible DNA damages.

Effects of desiccation on Euterpe edulis Martius seeds

Information on desiccation sensitivity of Euterpe edulis seeds under two drying rates is presented. The sensitivity was studied during the course of germination and normal germination. The water content was evaluated for both seeds and embryos. Results showed the following: (a) For both drying treatments and for both germination and normal germination, desiccation sensitivity values were higher for measurements based on the water content of the embryo than for those of the seed. (b) For both drying treatments, desiccation sensitivity were higher for normal germination than for germination based on both the embryo and seed water contents. (c) Under the slow drying treatment and for measurements based on the seed water content, critical water content was visible for normal germination but not for germination; (d) Critical water contents for germination and normal germination were more clearly established in the fast drying treatment than they were in the slow drying method based on both the embryo and seed water contents. Critical water contents were not associated with changes in electrolyte leakage, which suggests that conductivity is not a good indicator of physiological seed quality. From the beginning of both drying treatments, changes in nuclei and vacuoles were observed, but, when seed water content was reduced to below critical values, the cells became severely plasmolyzed, the vacuoles highly distorted, and the nuclei formed an almost homogeneous mass with the chromatin and the nucleoplasm, which suggests irreversible DNA damages.(AU)