制作活体草属虫临时装片的技巧

现介绍一种制作活体草履虫临时装片的技巧。１）用胶头吸管（长８０ｍｍ）吸取密度适当的草属虫液,将其１／４滴滴到载片中央,盖上盖片。２）取一张棱形的滤纸（对角线１．５ｃｍ长左右）用它的一个角尖触在盖片的一边,吸去多余的水分,稍候,再改用其另一个角尖继续吸,这样重复吸２～３次,即可将此装片用于实验观察,且效果十分理想。本方法优点：制片快。片中草履虫不易丢失;观察清楚,生动;制片成功率１００％;制片方法简便易行。制作活体草属虫临时装片的技巧@黄丽清$北京市日坛中学!100020     

γ-谷氨酰转肽酶活性电泳染色新方法

目的：建立一种新的电泳活性染色方法,以快速地对活性电泳中γ-谷氨酰转肽酶（GGT）进行显色,从而准确鉴定和定位该酶,并可用于该酶的电泳法纯化制备。方法：低温下,样品活性电泳结束后立即将浸有γ-L-谷氨酰-α-萘氨和双甘肽混合底物溶液的滤纸贴在胶面上反应5min,然后再用浸有对氨基苯磺酸和亚硝酸钠混合显色液的滤纸覆盖在基质滤纸上显色。结果：在滤纸上很快显示出一条红色条带,经切胶酶促反应检验确定为GGT,经电泳法和高效液相色谱法确定GGT达到了电泳纯。结论：该法快速、灵敏、简单、直观,为GGT的鉴定和纯化提供了一条新思路。     

人胃液乳酸脱氢酶同工酶定量电泳测定方法

本文介绍一个琼脂糖凝胶电泳定量测定胃液乳酸脱氢酶(LDH)同工酶的简易方法。包括一个灵敏的染色程序和光密度扫描,或洗脱比色。并完全适用于血清和组织抽提液中LDH同工酶分析。血清和组织提取液LDH同工酶的分离检测,最初用淀粉凝胶和琼脂凝胶电泳,四氮唑蓝(NBT)染色,以后用醋酸纤维薄膜电泳1961年提出的琼脂糖凝胶电泳,优于纸电泳和琼脂电泳的是:(1)无吸附作用,(2)含荷电基团少,电     

微波在蛋白质电泳染色和脱色中的应用

经过实验摸索和实践 ,我们发现一种利用微波处理在10ｍｉｎ内可使聚丙烯酰胺凝胶染色及脱色完成的方法 .ａ 将电泳后的凝胶 (厚度 0 75ｍｍ)取出 ,置于培养皿中用蒸馏水冲去残留缓冲液 .ｂ 加入染色液将凝胶完全浸泡其中 ,放入微波炉内高火档照射 2 0ｓ,停留 1ｍｉｎ ,再照射 10ｓ.ｃ 取出染色液 (回收还可再用 2次 ) ,用蒸馏水冲去凝胶上残留染色液 .ｄ 加入脱色液 ,高火档照射 2 0ｓ ,取出脱色液 ,然后加入新鲜脱色液再照射 2 0ｓ,重复5～ 6次 ,直至背景清晰透明 .实践中我们认为应注意以下几点 :ａ 盛试剂的培养皿上面应盖一稍大的培养…     

一种改进的双向电泳染色方法

本文涉及了双向电泳过程中的染色方法,即先用考马斯亮蓝染色,将胶上可见蛋白切下再银染的方法。这种方法可最大限度的减少胶中蛋白质点的损失,不仅避免了单一用考马斯亮蓝染色由于灵敏度不高而导致的低丰度蛋白的损失,也避免了单一用银染而使高丰度的蛋白因染色过度导致的损失。同时两种传统的染色方法结合完美,形成的新方法经济实用。     

陕西七纺器蛛和拉土蛛不同组织酯酶同工酶的比较研究

陕西七纺器蛛(Heptathela shaanxiensis),拉土蛛(Latouchia sp.)均于1987年10月采自陕西泾阳。在同条件下采用聚丙烯酰胺凝胶电泳技术进行了不同组织的酯酶同工酶比较。实验方法酶液的制备:将饥饿10天的两种蜘蛛按常规解剖,分别取各组织置于研钵内,根据样品重量之差异,分别加入40%蔗糖溶液1—3ml,匀浆过滤,离心(3500rpm×15′)。取上清液作酶源,贮存于4℃冰箱备用,点样时加入适量0.1%澳酚蓝作电泳的指示染料。酯酶同工酶的聚丙烯酰胺疑胶电泳分离和染色、脱色:基本参照邱琼华等(1987)的方法。用日本20M光密度计(波长570nm,狭缝0.4cm)扫描…     

一种产生纤维素酶菌种的筛选方法——染色滤纸条法

目前选育产纤维素酶菌种,一般初筛用滤纸溃烂法选取生长旺、使滤纸溃烂显著的菌株,作为初筛结果。复筛时制曲、萃酶、过滤、离心及活性测定等步骤,工作量大,效率低,影响菌种选育工作的进展。我们在选育纤维素酶菌种中,对国外新近介绍的染色滤纸条法稍作改进,对数百株真菌作了初筛和初步复筛,证明用染色滤纸条法,可减少工作量,并能以数百株菌同时投入初筛,提高了筛选效率,加速了菌种的选育过程。现将方法介绍如下。     

果蝇唾腺染色体的观察和制片方法的改进

几年来我们在教学中进行了多方面的实验探索,在果蝇唾腺制片方法上加以改进,采用加蔗糖液保色,树脂胶封边,制备一种半永久制片标本。操作过程如下: 1.取出唾腺,在其上加2滴(37℃)蒸馏水低渗处理10～20分钟,使唾腺细胞涨大有利于染色体的分散。 2.用滤纸吸去蒸馏水,加1滴1N HCl解离8～15分钟(时间随当时的温度而定),吸去解离液,用蒸馏水轻轻冲洗2～3次,将水吸净。     

SOD的聚丙烯酰胺梯度凝胶电泳分离和酶活性显示

SOD的聚丙烯酸胺凝胶电泳技术已被广泛应用于动、植物SOD同工酶的研究。我们改用浓度梯度胶电泳法分离和鉴定SOD,其谱带受杂蛋白干扰少,分辨率和灵敏度比均一胶电泳法高,并能根据电泳相对迁移率较为快速和准确地初步判定SOD同工酶类型。现简介如下：门）SOD浓度梯度胶的制作基本按张龙翔等方法（生化实验方法和技术,北京：人民教育出版社,1982．119）,凝胶梯度为4％～35％。制成的凝胶先预电泳除去残留的过硫酸按,点样后在4C左右电泳,一般SOD港带达到相对稳定需3500～4000V／h。（2）SOD活性染色按罗广华、王爱国方法［植…     

白及块茎铜,锌超氧物歧化酶的纯化及其性质

白及（Ｂｌｅｉｌｌａｓｔｒｉａｔａ（Ｔｈｕｎｂ．）Ｒｅｉｃｈｂ．ｆ．）的ＳＯＤ同工酶只有一条较宽的谱带,确认为Ｃｕ·Ｚｎ－ＳＯＤ。其块茎ＳＯＤ总活性和比活性都高,且含有丰富的白及胶;经丙酮分级沉淀,ＳｅｐｈａｄｅｘＧ１００凝胶过滤和ＤＥＡＥ－纤维素柱层析分离纯化,获得对ＣＮ￣－敏感的淡兰色Ｃｕ·ＺｎＳｏＤ粉末。在凝胶电泳染色图谱上,纯化后的酶与粗酶液的ＳＯＤ区带相对应,且其酶活性染色带与蛋白染色带位置对应,表明已纯化到均一程度。该酶分子量约３３ＫＤ,亚基分子量约为１６．４ＫＤ;紫外光区的吸收峰在２６４．６ｎｍ,等电聚焦电泳呈现一条蛋白区带,ｐＨ值在４．３５左右;该酶在ｐＨ６．０～１０．０,温度在５０℃范围内具稳定性。纯化后的酶为４５６３．２ｕ／ｍｇ·蛋白,纯化了５１倍,活力回收为２２．３％。上述酶没有过氧化氢酶活性。提取过程中还得到高质量的副产品白及胶。     

以靛酚乙酸酯为底物的酯酶同工酶显色方法的研究

建立一种以靛酚乙酸酯为底物的酯酶同工酶的显色新方法。酯酶样品的聚丙烯酰胺凝胶电泳（PAGE）凝胶用磷酸缓冲液漂洗约10min后,浸入含有0.002%靛酚乙酸酯的溶液显色5～10min,可显出清晰的蓝色酯酶带。先将酯酶凝胶板浸于有机磷农药溶液中,然后再用靛酚乙酸酯显色液显色,比较同工酶谱,从同工酶带由深蓝色变为浅蓝色的颜色变化,可以看出对有机磷农药敏感的同工酶所受到的抑制程度。     

A technique for detection and relative quantitative analysis of glucosephosphate isomerase isozymes from nanogram tissue samples

An improved method for detecting and measuring the enzyme glucosephosphate isomerase after starch gel electrophoresis is described. Nitrocellulose filters are used in a gel overlay procedure which increases the sensitivity of the staining reaction and provides a simple means for accurate quantitation of the isozyme pattern. This staining technique may have wider application with other gel media and also with other enzymes.This work was supported by the M.R.C. Group in Developmental Neurobiology, McMaster University, Canada.     

三种鼠科动物血浆超氧化物歧化酶和乳酸脱氢酶以及血浆蛋白成分的比较分析

本文利用SOD和LDH在一块凝胶板上同时显色的优点,电泳分析黑线姬鼠(Apodemus agrarius)、褐家鼠(Rattus norvegicus),黄胸鼠(Rattus flavipectus)血浆SOD和LDH同工酶以及血浆蛋白成分。结果表明,LDH为有色区带,SOD为无色透明区带,而且SOD比LDH泳动快,两类区带互不重迭,以致3种鼠血浆中SOD和LDH的图谱清晰可辨。由于黑线姬鼠的SOD和LDH的泳动度不同于其余2种鼠,周此3种鼠的图谱各具特征。3种鼠血浆蛋白中的前清蛋白和铁传递蛋白区带数和泳动度亦具有明显差异,说明上述酶分子亚基和血浆蛋白分子的化学结构不同,成为种间差异在分子水平上典型的表现。     

一种转移非特异性蛋白带的染色方法

考马斯亮蓝是常用的聚丙烯酰胺凝胶蛋白电泳的染料,利用硝酸纤维素膜(NCF)对染料的吸附作用,将低浓度的考马斯亮蓝(0.025%)染色液直接对NCF上的转移蛋白带进行染色,经实验反复验证.它是一种较好的NCF上转移非特异性蛋白带的染色方法.     

beta-Glucosidase of Penicillium funiculosum. II. Properties and mycelial binding

The properties of two isozymes of beta-glucosidase of Penicillium funiculosum (part I of this series) are described. The molecular weights of isozyme 1 was 2.3 x 10(5) by gel filtration and 1.2 x 10(5) by SDS gel electrophoresis, indicating two subunits. The molecular weight of isozyme 2 was unusually low for a fungal beta-glucosidase: 1.6 x 10(4) by gel filtration and 3.7 x 10(4) in the presence of isopropanol. The two enzymes differed from other fungal beta-glucosidases in their substrate specificities. They showed high activity with pNPG, cellobiose, cellotriose, cellotetraose, cellopentaose, gentiobiose, and laminarin, but were inactive with filter paper, CM cellulose, or derivatives or stabilized by bovine serum albumin and several alcohols such as butanol and propanol. It was inhibited by glucono-delta-lactone (K(i) = 0.67muM) and glucose (K(i) = 0.92mM).The enzyme was quantitatively adsorbed by P. funiculosum mycelium at pH 4 and the immobilized enzyme was as enzymically active as the free enzyme, but more heat stable. The binding efficiency was very high (5000 IU enzyme/g mycelium). It could be quantitatively eluted with buffers at pH 7 or by 0.02M Ca, Mg, or Al chlorides. The binding was selective, since mycelium grown on lactose could produce and also bind only beta-glucosidase isozyme 1, whereas mycelium grown on cellulose could produce as well as bind both beta-glucosidase isozymes as well as cellulases. Mycelial binding was unaffected by washing with EDTA or trypsinization, but was totally lost by washing with dilute KOH, HCl, or ethylenediamine.     

Isolation and characterization of two constitutive forms of microsomal cytochrome P-450 from a single bovine liver

Two constitutive forms of cytochrome P-450 isozyme were isolated from microsomes prepared from a single bovine liver. The two highly purified isozymes were electrophoretically homogeneous on SDS-polyacrylamide gel and their apparent minimum molecular weights were estimated to be 50 000 and 55 000. The isozyme of smaller molecular weight, designated cytochrome P-450A, and the one of large molecular weight, designated cytochrome P-450B, were distinct proteins by the criteria, SDS-polyacrylamide gel electrophoresis, peptide maps, amino acid contents. To reveal the immunochemical relation between these two isozymes, antibodies to each isozyme was raised in rabbit. Antibodies to cytochrome P-450A gave a single precipitin line against its antigen in Ouchterlony double-diffusion plates, but did not cross-react against cytochrome P-450B. On the other hand, antibodies to cytochrome P-450B formed a single precipitin line with its antigen and did not show any cross-reactivity against cytochrome P-450B. These results indicate that two isozymes are immunochemically distinct. This conclusion was supported by the results from immunochemical staining of the SDS-polyacrylamide gel electrophoretogram of the purified isozymes and detergent-solubilized bovine liver microsomes transferred to the nitrocellulose sheet. Both cytochromes P-450 showed high catalytic activities toward (+)-benzphetamine and aminopyrine in reconstituted systems, indicating that both enzymes have a high turnover number for N-demethylation.     

Characterization of the esterase isozymes of Ips typographus (coleoptera,scolytidae)

Four esterase isozymes hydrolyzing α-naphthyl acetate (α-NA) were detected screening whole body homogenates of larvae and adults of Ips typographus by electrophoresis. Two of the four isozymes (isozymes 3 and 4) were not detected by α-NA staining in the pupal stage, but topical application of juvenile hormone III (JH III) on the pupa induced these isozymes. The JH esterase (JHE) activity on the gel was associated with the proteins of isozyme 2. The compounds OTFP, PTFP, and DFP inhibited this catalytic activity of isozyme 2 on the gel at low concentrations, whereas the proteins of isozyme 3 and 4 were affected only at higher concentrations. A quantitative developmental study was performed to characterize which of the esterases hydrolyzed JH III, using a putative surrogate substrate for JH (HEXTAT) and α-NA. The I₅₀ of several esterase inhibitors and the JH metabolites were also defined. All findings supported the results that a protein associated with isozyme 2 is catabolizing JH and that isozymes 3 and 4 are the main contributors to the general esterase activity on α-NA. The JHE from Tenebrio molitor was purified by affinity chromatography. Although the recovery was low, an analytical isoelectric focusing gel showed that the JHE activity of the purified enzyme. T. molitor cochromatographed at the same pl as the JHE activity of I. typographus. Arch. Insect Biochem. Physiol. 34:203–221, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of lactate dehydrogenase enzyme in seminal plasma of Japanese quail (Coturnix coturnix japonica)

Lactate dehydrogenase enzyme present in quail seminal plasma has been characterized. Polyacrylamide gel electrophoresis and subsequently with LDH specific staining of seminal plasma revealed a single isozyme in quail semen. Studies on substrate inhibition, pH for optimum activity and inhibitor (urea) indicated the isozyme present in the quail semen has catalytic properties like LDH-1 viz. H-type. Furthermore, unlike other mammalian species, electrophoretic and kinetic investigations did not support the existence of semen specific LDH-X isozyme in quail semen. The effect of exogenous lactate and pyruvate on sperm metabolic activity was also studied. The addition of 1 mM lactate or pyruvate to quail semen increased sperm metabolic activity. Our results suggested that both pyruvate and lactate could be used by quail spermatozoa to maintain their basic functions. Since the H-type isozyme is important for conversion of lactate to pyruvate under anaerobic conditions it was postulated that exogenous lactate being converted into pyruvate via LDH present in semen may be used by sperm mitochondria to generate ATP. During conversion of lactate to pyruvate NADH is being generated that may be useful for maintaining sperm mitochondrial membrane potential.     

Alcohol dehydrogenase loci of sterile and fertile lines of tomato in the process of ontogenesis

A biochemical analysis through electrophoresis in poly-acrylamide gel of alcohol dehydrogenase loci in three ontogenetic phases of tomatoes’ development was carried out. This subject of investigation were the male-sterile line 6944 aa and the fertile anthocyan line 6944 aa⁺. Higher levels of isozyme expression in leaves in the cotyledon phase of anthocyan plants, particularly of isozyme No 1 in the Adh-2 locus, was found. The connection between alcohol dehydrogenase, and pollen male sterility and anthocyan staining (aa⁺) were discussed. A quantitative molecular marker connected to a genetic marker (in this case) for the purposes of identifying sterile from fertile plants in the earliest phase of their development was established.     

Induction of manganese peroxidase and laccase by <Emphasis Type="Italic">Lentinula edodes</Emphasis> under liquid culture conditions and their isozyme detection by enzymatic staining on native-PAGE

The white-rot basidomycete Lentinula edodes often produces the lignin-degrading enzymes manganese peroxidase (MnP; EC 1.11.1.13) and laccase (Lcc; EC 1.10.3.2) in sawdust-based media. In the present study, MnP from L. edodes was induced under liquid culture supplemented with sawdust extracts of Castanopsis cuspidata. Lcc activity was induced by the addition of 2 mM CuSO₄·5H₂O into the same media 7 days after initial inoculation. Phenoloxidase enzymes were distinguished by polyacrylamide gel electrophoresis (native-PAGE), followed by sequential enzymatic staining with an improved staining solution. The isozyme bands detected under MnP-induced conditions were identified as manganese peroxidase (lemnp2) and bands detected under Lcc-induced conditions were identified as laccase (lcc1) by Q-TOF mass spectrometry.