A comprehensive evaluation of imidazole‐zinc reverse stain for current proteomic researches
Ching‐Yu LinVinchi WangHao‐Ai ShuiRong‐Huay JuangAi‐Ling HourPei‐Sing ChenHuiming HuangSzu‐Yu WuJen‐Chieh LeeT Y TsaiHan‐Min Chen
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Abstract:
In this paper, we comprehensively evaluated the capability of imidazole-zinc reverse stain (ZN) in comparative proteomics. Three commonly used protein gel staining methods, including silver (SN), SYPRO Ruby (SR), and CB stain were investigated alongside for comparison purpose. A transparency scanning procedure, which may deliver more even and contrasting gel images, was found best for documenting ZN stained gels. Our results showed that ZN was more sensitive than SN, SR, and CB. It may reveal as few as 1.8 ng of proteins in a gel. Moreover, ZN was found to provide a linear dynamic range of staining for revealing proteins up to 140 ng, and show an insignificant staining preference. To analyze a ZN stained 2-D gel image that generally comprises an apparent but even background, the Melanie 4 software was found more suitable than others. Furthermore, ZN demonstrated an equivalent or better MS compatibility than the other three staining methods. Intense and comprehensive MS profiles were frequently observed for ZN stained gel spots. Approximate two-third of ZN stained gel spots were successfully identified for protein identities. Taken together, our results suggest that the prompt, cost effective and versatile ZN is well suited for current proteomic researches.Keywords:
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To enhance the efficiency of silver staining and band recovering in Polyacrylamide Gel Electrophoresis(PAGE),both conventional silver staining and speedy silver staining were investigated,using the technique of ISSR-PCR markers with Magnolia grandiflora DNA.During band-recovering,conventional ethanol hepatin method and improved boiling method were employed and followed by re-amplification of PCR identification.The results indicate that the two methods produce similar clear bands and band patterns,but the procedure of conventional silver staining takes 2～3 hours while the speedy silver staining only requires 30 minutes which reduces the experimental time by 4～6 times.The improved boiling method is more convenient and time-saving than the conventional one.Speedy silver staining and improved boiling band-recovering can be employed in the future studies.
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—InThe Journal, April 14, p. 1092, Dr. Peter Mjedloff requests some further information concerning a bacillary stain in general use at the Kula Sanitarium. The stain to which he refers apparently is the stain designated as Much's granular stain. The stain which is in use at the sanatorium is identical with Much's stain, differing, however, in the fact that the Lugol's solution is used first on the prepared smear and this is then followed by the gentian-violet stain. After destaining with acid in the usual way, counterstaining in Much's technic is by means of a 1 per cent. methylene blue solution, although any other counterstain or dye, such as pyronin, may be used. In the usual staining technic this method of staining the tubercle bacilli is not considered superior to the carbolfuchsin or Ziehl-Neelsen method for detecting the bacilli if in their integrity. It is onlyStain
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Differential staining
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Objective: To compare the effect and their sensitivity of two different staining methods to DNA in the gels, and evaluate the possible application of SYBR GreenⅠin routine DNA staining as a substitute for ethidium bromide(EB). Methods: The new nucleic acid staining SYBR GreenⅠand EB were used to stain the DNA in the gel, perfectively stain before electrophoresis and after electrophoresis. Results: There was no distinct difference between the two methods. Both SYBR GreenⅠand EB stain could detect as little as 10 ng per band of DNA. Conclusion: The effect and sensitivity of the two staining methods are no distinct difference, and SYBR GreenⅠand EB may be used in these staining methods. SYBR GreenⅠ, which is a new nucleic acid stain may substitute for EB as a general dyestuff to stain the DNA in the gel.
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A simple method was described for negative gold staining of ultrathin-layer polyacrylamide gels. The protein bands were clear (unstained) and sharp while the gel matrix was pink-purple. Negative gold staining was up to five-fold more sensitive than coomassie blue, but an order of magnitude less sensitive than silver staining for protein detection. However, there were examples where negative gold staining was able to detect particular proteins not effectively stained by coomassie blue or silver.
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AbstractThe importance of pH in staining tissue is emphasized. The effect of pH upon the selectivity and intensity of staining with iron hematoxylin, malachite green, and eosin Y is considered. Many difficulties may be avoided by staining in the higher alcohols and directions are given for the preparation of buffer solutions from pH 1.2-8 in alcohol. The concentration of stains, time of staining, and order of staining are discussed for progressive and regressive staining. At pH 8 in 95% alcohol very few tissues stain with malachite green at a concentration of 1/1000 saturated. At pH 6 most cytoplasmic elements stain with malachite green at a concentration of 1/1000 saturated or with eosin Y at 1/250 saturated. As the pH is lowered more tissue elements stain until the nucleus is completely stained. This behavior is in accord with the theory of chemical combination of dyes with proteins, which states that proteins combine with basic dyes on the basic side of their isoelectric points and with acid dyes on the acid side of their isoelectric points. With hematoxylin stain the pH range is much shorter. A satisfactory hematoxylin stain is composed of 0.1% hematoxylin, 0.1% FeCl3, and HCl to bring the pH to 1.2-1.6 in 80% alcohol. With this stain, which may be used immediately, the nuclei of most tissues begin to stain at pH 1.2 and much of the cytoplasm will be stained if the pH is raised to 1.4. The shortness of this effective pH range is thought to be due to the dissociation of the hematoxylin-iron-protein complex. The use of different dyes successively at different pH values, such as hematoxylin at 1.3, malachite green at 8, and eosin at 6, permits better differentiation of the tissue elements, and intelligent variations in the staining technic.
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Many polychromatic stains are in use for epoxy-embedded tissues (Horobin 1983). We should like to report a quick and easy polychromatic staining procedure that we find useful for routine use. Formerly the stain we used was prepared in 20 ml water and 5 ml 96% alcohol, and gave polychromatic staining only at pH 7.4 obtained by the addition of 1 N NaOH. However, the stain gave satisfactory results only for two or three days. We found that stabilization of the staining solution through the use of an ethyl alcohol-cacodylic buffer solvent increased the life of the staining solution. This was convenient because the cacodylic buffer is used in our laboratory as a component of fixatives, and is not prepared specially for the staining.
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Negative staining is one of the many staining techniques that can be employed for viewing of bacterial cell morphology and size. The advantages of the negative stain include the use of only one stain and the absence of heat fixation of the sample. Negative staining employs the use of an acidic stain and, due to repulsion between the negative charges of the stain and the bacterial surface, the dye will not penetrate the cell. In negative staining, the results yield a clear cell with a dark background.
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A simple and well reproducable modification of the Papanicolaou stain is reported. The most important modification is the replacement of the natural stain hematoxyline by the qualitively excellent synthetic stain thionin which is immediately added to the alcoholic fixation solution for a fixation stain. The high labor intense alternation between alcohol water and alcohol is thus eliminated. The quality of the nuclear staining is very good. The fixation staining with thionin without counter-staining of the cytoplasma may be used for a rapid thionin stain which gives sufficient cytoplasmatic staining by the thionin for a rapid cytological smear for cancer. The counter-stain of the cytoplasma was also simplified. The over-all staining of this simplification of the pap smear is comparable to that of the original papanicolaou stain. This modification of the papanicolaou stain fulfills requirements which a stain in competition with the original papanicolaou stain should have. 1. The modification is qualitively equal to the original method, 2. the staining method is simpler, 3. the staining method is well reproducable, 4. the staining method is more economical.
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