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Results for “protocol optimization”

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PubMed2003

Modelling a minimal residual disease-based treatment strategy in childhood acute lymphoblastic leukaemia.

The measurement of minimal residual disease (MRD) in childhood acute lymphoblastic leukaemia offers the promise of individualized, risk-stratified treatment, but an optimal protocol needs establishing. A model was developed to explore certain unanswered questions. The model assumes that all patients have MRD assessed after induction chemotherapy and children above a certain threshold are offered intensive chemotherapy. Using parameter estimates derived from published studies of MRD, the model predicted event-free survival (EFS) rates, relapse rates and treatment-related mortality for a cohort of children in the first presentation who were Philadelphia chromosome negative. Using the level of MRD after induction in order to decide on the use of intensive therapy resulted in an increase in EFS rates of up to 2.9 per 100 children, although if the error of the MRD measurement were too great, the benefit was almost nullified. Taking and analysing more than one marrow sample from the patient for the MRD measurement, in order to reduce sampling and measurement error, improved EFS by a further 1.1 patients per 100 treated, and decreased the number of patients offered intensive therapy by up to 2.6 per 100 treated. The optimal threshold for offering intensive therapy was in the range of 10-3.5- 10-4.5 cells if the intensive treatment-related mortality was 13-18% (allograft options), but 10-5- 10-6 cells if it was less than 8% (intensified chemotherapy). Using MRD to target patients at a high risk of relapse improved EFS rates, but the accuracy of measurements was of critical importance.

Antineoplastic Agents↗

PubMed1996

Group B streptococcus and pregnancy.

Group B streptococcus is a significant pathogen for both mother and child. routine urine culture in pregnancy will identify and allow treatment of women with asymptomatic bacteriuria. An optimal protocol for the prevention of neonatal sepsis has not yet been developed. While intrapartum antimicrobial prophylaxis appears to provide the best potential, each of the currently suggested protocols has significant drawbacks. Drawbacks include the potential for missing high-risk carriers, failure to treat a significant proportion of those destined to deliver an affected infant because no risk factors are present, and empirical treatment of a large proportion of the population in order to present significant disease in a few. Until an effective program of immunization becomes available, intrapartum prophylaxis of group B streptococcal carriers appears to offer the best hope of reducing the incidence of neonatal disease. Caregivers should adopt a uniform practice with regard to screening and prophylaxis. It is essential that any broad-based screening program include an evaluation of efficacy as well as complications including the development of new etiologic agents as causes of neonatal sepsis and the emergence of resistant bacteria. Further, mothers and newborns should be evaluated for drug adverse reactions and the impact of intrapartum prophylaxis on the use of prolonged empirical broad-spectrum antimicrobial therapy on the asymptomatic infant. Additional research is necessary regarding the required duration of therapy for optimal effect of intrapartum prophylaxis, the need for postnatal prophylaxis of the asymptomatic neonate, and the optimal agent for neonatal prophylaxis (penicillin versus broad-spectrum agents) if neonatal therapy is necessary after intrapartum prophylaxis.

Female↗

PubMed2003

[Endoluminal diode laser vein damage: preclinic study for vein insufficiency].

BACKGROUND: The ex-vivo phase of this research aims to define fibre laser activity ranges with the intent of using a safety protocol on the operating table to ensure maximum results in occluding the vein lumen with minimum necessary energy parameters. METHODS: The study was performed on 20 human saphenous veins removed during phlebological surgery. A 1 mm diameter optical fibre was inserted in the vein to the proximal extremity where administration of laser energy began along the vein axis. The veins were subdivided into three groups based on the parameters used: Group 1: treated with 20 Watt power; Group 2, treated with 15 Watt power; Group 3, treated with 10 Watt power. On conclusion of the procedure the veins were immersed in formalin and sent for histological examination. RESULTS: The greatest endothelial damage was observed in the group of veins treated with fluence above 300 J/cm(2), in which carbonisation of the endothelium was observed; in those treated with lower fluence (200-100 J/cm(2)) damage consisted in coagulation necrosis and interstitial oedema; at fluence of 100 J/cm(2) endothelial damage was less uniform. CONCLUSIONS: Experimental evidence would appear to support the hypothesis that the optimal protocol is below 300 J/cm(2) fluence with 15 Watt power and pulse train 100-300 ms.

Adult↗

PubMed2005

Optimization of the differentiation of human preadipocytes in vitro.

This study aimed at developing an optimal protocol for proliferation and differentiation of preadipocytes that is a prerequisite for constructing an ideal biohybrid composed of viable adipose precursor cells in a three-dimensional matrix. Such an implant could represent an adequate solution for correcting soft tissue defects, e.g., extensive deep burns or tumor resections. Preadipocytes were isolated from human subcutaneous adipose tissue samples and cultured in Dulbecco's modified eagle medium (DMEM)/Ham's F12 medium (F12) or OPTIMEM medium with or without the addition of human serum (hS) or fetal calf serum (FCS). The advantages of fibronectin-coated culture dishes for preadipocyte yield after isolation and differentiation were evaluated. After culture expansion, differentiation was induced by insulin, isobutylmethylxanthine, pioglitazone, dexamethasone, and transferrin in the absence of serum. The extent of differentiation was assayed by measuring the activity of glycerophosphate dehydrogenase as well as counting of differentiated versus undifferentiated cells. Our results show that fibronectin coating does not only strongly increase the yield of preadipocytes after isolation from adipose tissue but also significantly enhances differentiation of precursor cells to mature adipocytes. For optimal cell expansion, DMEM/F12 is more promoting than OPTIMEM and culturing with FCS shows a slightly better proliferation compared with hS supplementation. Differentiation, in contrast, is significantly improved when hS is used instead of FCS during proliferation. Our results smooth the way for autologous preadipocyte culturing and show that hS for preadipocyte culturing opens new and promising perspectives for adipose tissue engineering by optimizing in vitro expansion in cell culture and inducing substantial differentiation.

Adipocytes↗

PubMed2005

Optimization of expression and purification of two biologically active chimeric fusion proteins that consist of human interleukin-13 and Pseudomonas exotoxin in Escherichia coli.

We have previously reported that a variety of solid human tumor cell lines express a large number of receptors for interleukin-13 (IL-13). These receptors could be targeted with a chimeric fusion protein consisting of human IL-13 and a truncated form of Pseudomonas exotoxin (PE). We describe here optimization of critical steps involved in high yield expression of two recombinant chimeric fusion proteins for obtaining highly purified and biologically active cytotoxins in Escherichia coli. The chimeric constructs of human IL-13 and two 38 kDa truncated PEs: (i) PE38 and (ii) PE38QQR, (three lysine residues in PE38 at 590, 606, and 613 substituted with two glutamine and one arginine) were used for protein expression in pET prokaryotic expression vector system with kanamycin as a selection antibiotic. Our results suggest that fresh transformation of E. coli and induction by isopropyl-beta-D-thiogalactopyranoside (IPTG) for 6 h resulted in maximum protein expression. To further improve the yield, we used a genetically modified E. coli strain, BL21(DE3)pLysS, which carries a plasmid for lysozyme with a weak promoter that inhibits T7 RNA polymerase and minimizes protein production in the absence of IPTG. Use of this strain eliminated the need for lysozyme digestion of the induced bacteria to release inclusion bodies, which resulted in expression of purer protein as compared to the conventional BL21(DE3) strain. Additional protocol optimizations included 16 h solubilization of inclusion bodies, constitution of refolding buffer, and timing of dialysis. These proteins were finally purified by Q-Sepharose, mono-Q, and gel filtration chromatography. Between 14-22 and 21-28 mg highly purified and biologically active protein was obtained from 1L of BL21 (DE3) and BL21 (DE3) pLysS bacteria culture, respectively. As IL-13R targeting for brain tumor therapy offers an exciting treatment option, optimization of production of IL-13PE will enhance production of clinical grade material for Phase III clinical trials.

ADP Ribose Transferases↗

PubMed2004

Interlaboratory comparison of fetal male DNA detection from common maternal plasma samples by real-time PCR.

BACKGROUND: Analysis of fetal DNA from maternal plasma by PCR offers great potential for noninvasive prenatal genetic diagnosis. To further evaluate this potential, we developed and validated a standard protocol to determine whether fetal DNA sequences could be reproducibly amplified and measured across multiple laboratories in a common set of specimens. METHODS: Each of five participating centers in a National Institute of Child Health and Human Development consortium collected 20 mL of peripheral blood from 20 pregnant women between 10 and 20 weeks of gestation. The plasma fraction was separated according to a common protocol, divided, and frozen in five aliquots. One aliquot was shipped to each participating laboratory, where DNA was extracted according to a standard protocol. All plasma samples (n = 100) were then analyzed blindly for the presence and quantity of total DNA (GAPDH) and male fetal DNA (SRY) by real-time PCR. Genomic DNA was isolated from female and male cells at one center, quantified, and shipped to the others to serve as calibrators for GAPDH and SRY, respectively. RESULTS: The amplification of known quantities of DNA was consistent among all centers. The mean quantity of male DNA amplified from maternal plasma when the fetus was male ranged from 51 to 228 genome equivalents (GE)/mL. Qualitative concordance was found overall among centers. The sensitivity of the assay for detection of male DNA when the fetus was male varied from 31% to 97% among centers. Specificity was more consistent (93-100%) with only four false-positive results obtained across the entire study. CONCLUSIONS: All centers were able to consistently amplify frozen and shipped DNA. The PCR procedure used here is reliable and reproducible. Centers that extracted and amplified more DNA per milliliter of maternal plasma had superior sensitivities of Y chromosome sequence detection. The specificity of the assay was more consistent among centers. A robust and thoroughly optimized protocol for the extraction of DNA from maternal plasma is needed to make testing of fetal DNA in maternal plasma a clinically relevant analytical tool.

Clinical Laboratory Techniques↗

PubMed1998

Incidence and impact of Clostridium difficile infection in the UK, 1993-1996.

Questionnaires were sent to 360 UK medical microbiologists to determine the incidence of Clostridium difficile infection in the UK between 1993-1996, and to establish the current laboratory testing protocols. Replies were received from 104 laboratories (29% response rate), 86, 7, 4 and 3% of which are in England, Scotland, Wales and Northern Ireland, respectively. The laboratories serve a total of approximately 90,000 hospital beds (median 750). C. difficile testing was performed by 83% of the laboratories, and 52, 45 and 31% used toxin A +/- B kits, cell cytotoxicity, and culture +/- isolate toxigenicity testing methods, respectively. Forty-seven percent of laboratories only performed testing when specifically requested, 19% if antibiotic use was stated, 15% tested all diarrhoeal specimens, and 14% examined all specimens except those from infants and community patients. The annual totals of positive C. difficile reports and cases increased from 3132 to 12,775, and from 1576 to 8211, respectively, between 1993-1996. In 1993 C. difficile infection caused ward closures in 5% of hospitals but 16% in 1996. Antibiotic policy changes, due to C. difficile infection, occurred in 21% of hospitals in 1996 compared with only 4% in 1993. C. difficile infection appears to be increasing markedly in the UK with major implications on hospital inpatient activity. Wide variations in laboratory selection and testing methods are likely to be masking the true epidemiology of C. difficile infection, and consensus is required on optimal protocols.

Aged↗

PubMed1997

Induction of superovulation in cyclic rats by administration of decreasing doses of recombinant follicle stimulating hormone (Org32489).

The objective of this study was to set up a superovulation protocol in adult cyclic rats by using recombinant human follicle stimulating hormone (rhFSH; Org32489). Good results were obtained by treatment with decreasing doses of rhFSH (2.5 to 0.5 IU) during the dioestrus period. The number of corpora lutea (CL) found in rats treated with this protocol was 43.5 +/- 3.4; this is more than three times the number in saline-treated control rats (13.0 +/- 0.4). Fertilization of oocytes after superovulation was as good as after normal ovulation in terms of number of 2-cell stage embryos found 2 days after mating. The absolute number of implantations was twice the number observed in saline-treated control rats (23.3 +/- 1.8 versus 10.6 +/- 0.5); therefore the number of implantations per CL was lower in superovulated rats. The serum concentrations of luteinizing hormone (LH), endogenous FSH and oestradiol-17beta were decreased during rhFSH treatment, while the inhibin serum concentration was increased. The progesterone serum concentration was increased on the days of pro-oestrus and oestrus after treatment. No difference was observed in the testosterone serum concentration. Pretreatment with 10 IU rhFSH at oestrus before giving the decreasing doses of rhFSH during dioestrus reduced the ovulatory response. Finally, treatment with a constant low dose of rhFSH instead of a decreasing dose of rhFSH did not result in spontaneous ovulation. However, ovulation induction by means of a human chorionic gonadotrophin bolus resulted in superovulation in six out of eight rats. It is concluded that superovulation in cyclic rats can be achieved using rhFSH treatment. However, it was found that the type of rhFSH regimen was very important to achieve appropriate stimulation. The optimal protocol was treatment with decreasing doses of rhFSH during dioestrus. The oocytes retrieved could be fertilized as well as oocytes of saline-treated control rats. The results also indicate that treatment with higher doses of rhFSH might induce a desensitization for FSH and LH.

Animals↗

PubMed1996

Temporal and molecular characteristics of lacZ mutations in somatic tissues of transgenic mice.

In order to help establish criteria for optimizing protocols for in vivo mutation studies, lacZ transgenic mice (Muta mouse) were treated with five consecutive daily doses of ethylnitrosourea (50 mg/kg), sampled at times up to 55 days after treatment, and mutant frequencies and DNA sequences determined for liver and bone marrow. In the bone marrow, the mutant frequency rose very rapidly in the first 5 days after treatment to 34 times the control frequency. Subsequently, there was a brood peak where the mutant frequency did not vary significantly, although it did appear to begin to decline after 45 days. In contrast, in the liver, the peak mutant frequency (11 times the control frequency) was not achieved until 35 days, after which there appeared to be a slow decline up to 55 days, which was not statistically significant. Once the maximum mutant frequency was reached, the mutation spectra in the two tissues were indistinguishable. In contrast to the G:C-->A:T transitions in 5'-CpG sites characteristic of untreated mice, A:T-->T:A transversions and A:T-->G:C transitions were prominent in both liver and bone marrow of ENU-treated mice, suggesting the involvement of unrepaired O2- and O4-ethylthymine adducts. In addition, G:C-->T:A transversions were induced in liver. This study demonstrates the possibility that although tissues may have different mutation fixation times, a single mutation fixation time equal to the longest time may be appropriate for in vivo mutation studies, provided that the mutation frequency does not decline appreciably after the peak is reached. This study also illustrates the necessity of ensuring that mutation characteristics are determined after optimal fixation has occurred.

Animals↗

PubMed1998

Different modalities of spa therapy for skin diseases at the Dead Sea area.

BACKGROUND: Balneology and spa therapy, although not accepted as well-established treatment modalities in dermatology, are used throughout the world. The therapeutic properties for skin and rheumatic diseases of the Dead Sea area may be attributed to unique climatic characteristics and unique natural resources. The mechanisms by which a broad spectrum of diseases are alleviated by spa therapy may involve mechanical, thermal, and chemical effects. OBJECTIVE: To review and discuss various spa therapy modalities, used at the Dead Sea area for a wide spectrum of skin diseases. CONCLUSIONS: Existing evidence indicates the therapeutic potential of Dead Sea spa therapy modalities for psoriasis and psoriatic arthritis. A beneficial effect is hinted at for other skin diseases, but the absence of relevant methodological and clinical information precludes the drawing of any scientific conclusions. It is essential to establish therapeutic guidelines to determine the optimal treatment modality for each disease, and the optimal protocol of each treatment component, adjusted individually for each patient, with respect to remission and long-term adverse effects.

Balneology↗

PubMed1999

Dual colour fluorescence in situ hybridization to paraffin-embedded samples to deduce the presence of the der(X)t(X;18)(p11.2;q11.2) and involvement of either the SSX1 or SSX2 gene: a diagnostic and prognostic aid for synovial sarcoma.

Identification of the t(X;18)(p11.2;q11.2) and the fusion gene products, SYT-SSX1 and SYT-SSX2, associated with a high proportion of synovial sarcomas, has been shown to be a useful diagnostic aid. This study demonstrates the application of dual colour fluorescence in situ hybridization to paraffin-embedded samples to deduce the presence of the derivative X chromosome and also the position of the breakpoint on chromosome X at either the SSX1 or the SSX2 gene. This used region specific markers from chromosomes X and 18 and an optimized protocol involving microwave exposure. Novel and rapid scoring criteria were validated which circumvented potential problems of nuclear truncation and defining cell boundaries. This involved blind analysis of two negative sarcoma samples and three synovial sarcomas in which corresponding frozen material had been previously shown to have the translocation involving different SSX genes. Six new cases diagnosed as synovial sarcoma were also analysed; two monophasic and two biphasic case were deduced to have a breakpoint in the SSX1 gene, one monophasic case an SSX2 breakpoint, and one case did not show rearrangement of the region. The ability to analyse formalin-fixed, paraffin-embedded samples in this way has practical implications for aiding the diagnosis of difficult cases, recently ascribed prognostic relevance, and allows further retrospective studies to be carried out. The methodology is also applicable to the identification of other tumour specific translocations in paraffin-embedded material.

Adult↗

PubMed1999

Electrohydrodynamically induced aggregation during constant and pulsed field capillary electrophoresis of DNA.

We present a study aimed at understanding the factors affecting the separation of large DNA molecules by capillary electrophoresis in polymer solutions. In a first series of experiments, a systematic study of the effect of operational parameters on the development of an electrohydrodynamic instability resulting in DNA aggregation and spurious peaks in the electropherograms is presented. The results are discussed in regard to a recent theory of electrohydrodynamic instabilities in macroion suspensions, recently proposed by Isambert et al. Overall, the results provide strong support to the theory. Some situations of interest for applications, and not explicitly considered in the theory, such as asymmetric field pulsing and the use of polymer additives in the buffer, were also considered. Furthermore, robust optimized protocols for high resolution separation of DNA in the range of 100 base pairs to 160 kilobase pairs, are proposed. As predicted by the model, it is shown that using a concentrated isoelectric buffer (histidine) strongly reduces aggregation as compared to the use of a conventional buffer at the same concentration, and allows separation of DNA from 100 bp to 160 kbp in less than 10 min. We also present a systematic study of the dependence of the mobility vs DNA size, pulse frequency, and field strength. The results are discussed with respect to the Biased Reptation with Fluctuations model and a good agreement is obtained. Copyright 1999 John Wiley & Sons, Inc.

Journal Article↗

PubMedDate not supplied

Concentration of viral vectors by co-precipitation with calcium phosphate.

BACKGROUND: The envelope glycoproteins, surface unit (SU) and transmembrane (TM) of the murine leukemia virus (MLV) are not covalently linked and tend to dissociate upon high-speed centrifugation, leading to loss of vector infectivity. This study describes a gentle and simple method to concentrate MLV vectors or HIV vectors pseudotyped with MLV envelopes. Having a fast and inexpensive method to concentrate large volumes of vector supernatant will facilitate in vivo experiments and clinical trials that require high titer vector stocks. METHODS: The methods employed in the study were co-precipitation of viral supernatant with calcium phosphate, low-speed centrifugation, dialysis, and infection assays with Lac-Z transducing vectors. RESULTS: Murine leukemia virus vectors and HIV vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV.G) or MLV envelopes were concentrated successfully using the calcium phosphate co-precipitation method. Parameters that influence virus yield and the reproducibility of the method were investigated. The optimized protocol involves virus harvest in serum-free media, co-precipitation using 60mM calcium chloride, pelleting at 2,000 g, resuspending the pellet in a small volume of 0.1M EDTA-saline, and dialysis against saline to remove EDTA. Volumes were decreased from 300 ml to 10 ml, with 50-100% recovery, and titers can be concentrated up to 1,000-fold. CONCLUSIONS: The calcium phosphate co-precipitation method to concentrate virus is applicable to retrovirus and lentivirus preparations. It uses simple techniques and does not require expensive equipment. Multiple rounds of co-precipitation can be carried out if required.

Animals↗

PubMed2002

Multiwell in-gel protein digestion and microscale sample preparation for protein identification by mass spectrometry.

In-gel peptide digestion has become a widely used technique for characterizing proteins resolved by two-dimensional gel electrophoresis. Peptides generated from gel pieces are frequently contaminated with detergent and salts. Prior to matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis, these contaminants are removed using micro scale C18 sample preparation columns. In this paper, data are presented to demonstrate the application of a solvent resistant MultiScreen 96-well plate with a low peptide binding membrane and ZipTip micropipette based sample preparation. Recoveries of peptides (m/z of 1000 to 5000 Da) derived from standard protein protease digests, were estimated at various stages of the analytical process. An optimized protocol has been established and all the reagents and consumables have been packaged in a ready to use commercial kit. Data will be presented to show the application of this technology package to accelerate the throughput of protein characterization by protease fragmentation.

Blood Protein Electrophoresis↗

PubMed2002

Bioprocess applications of a Sindbis virus-based temperature-inducible expression system.

The production and study of toxic proteins requires inducible expression systems with low basal level expression and high inducibility. Here, we describe bioprocess applications of the pCytTS temperature-regulatable Sindbis virus replicon-based expression system. We used green fluorescent protein as a marker protein to optimize the selection of stable transfected clones with increased expression levels. Using the optimized protocol, clones were constructed that produced the growth-inhibiting, anti-viral protein interferon beta (beta-IFN). Selected clones were analyzed for temperature-dependent beta-IFN production in adherent and suspension cultures in serum free medium. Specific expression levels were around 1.0 x 10(5) IU/10(6) cells/day (0.5 microg/10(6) cells/day) in suspension cultures and over 1.5 x 10(6) IU/mL/day (7.5 microg/mL/day) in hollow fiber reactors using adherent cells. Hexahistidine-tagged beta-IFN purified from T-flask cultures was highly glycosylated and showed high specific activity. beta-IFN mRNA amplified by the viral replicase for 10 days did not show an accumulation of mutations. These data suggest the applicability of the pCytTS-inducible expression system for the production of high-quality glycoproteins in different reactors.

Animals↗

PubMed2004

Optimized conditions for diluting and reusing a fluorescent protein gel stain.

This study elucidates the optimum conditions at the minimum cost for using SYPRO Ruby protein gel stain. It deals with the effects of gel fixation and staining times, as well as dilution and reuse of SYPRO Ruby protein gel stain in one-dimensional (1-D) gels. Signal strength and dynamic range were highest in gels that were fixed thoroughly before staining, followed by overnight staining. Using the optimized protocol, dilution or reuse of the stain reduces the dynamic range and signal intensity. Sensitivity remains high if the stain is reused up to two times, but signal intensity is reduced up to 2.5-fold in twice used stain. Sensitivity also remains high if the stain is diluted 1:2 in water, but signal intensity is reduced up to 6-fold. Of the two options, reuse or dilution, reuse better retains signal intensity and dynamic range.

Electrophoresis, Polyacrylamide Gel↗

PubMed2006

A chip-based miniaturized format for protein-expression profiling: the exploitation of comprehensively produced antibodies.

Numerous antibodies have been developed and validated in recent years, and show promise for use in novel functional protein assays. Such assays would be an alternative to pre-existing comprehensive assays, such as DNA microarrays. Antibody microarrays are thought to represent those functional protein assays. While a variety of attempts have been made to apply DNA microarray technology to antibody microarrays, a fully optimized protocol has not been established. We have been conducting a project to comprehensively produce antibodies against mouse KIAA ("KI" stands for "Kazusa DNA Research Institute" and "AA" are reference characters) proteins. Using our library of antibodies, we established a novel antibody microarray format that utilizes surface plasmon resonance (SPR) technology. A label-free real-time measurement of protein expression in crude cell lysates was achieved by direct readout of the bindings using SPR. Further refinement of the antibody microarray format enabled us to detect a smaller quantity of target proteins in the lysate without the bulk effect. In this review, we first summarize available antibody array formats and then describe the above-mentioned format utilizing updated SPR technology.

Animals↗

PubMed2007

Simultaneous detection of molecular weight and activity of adenylate kinases after electrophoretic separation.

Adenylate kinases (AKs) are ubiquitous monomeric phosphotransferases catalyzing the reversible reaction, AMP + MgATP = ADP + MgADP, which plays a pivotal role in the energetic metabolism. In vertebrates, six AK isoforms are known. In this work, we report the detection of many AK isoforms directly on gel or NC after separation by denaturing electrophoresis and electroblotting, by an optimized protocol for the enzyme detection. The method allows to clarify the apparent MW of most of those AK isozymes that follow the cited reaction, especially onto NC where bands are sharper due to the absence of protein diffusion. In contrast, GTP:AMP phosphotransferases are not detectable. AK activity from many sources can be detected in both its reaction courses; ATP production appears as dark-blue bands, while ADP formation appears as nonfluorescent bands over a fluorescent background, under long-wavelength UV light. We show that nondenaturing gel electrophoresis is not the first choice for AK activity detection. Our method is different from the preceding reports on AK activity detection in bacteria after native polyacrylamide gel separations, in the absence of SDS or methanol. The procedure is also quantitative, allowing to determine the amount of enzyme present in samples.

Adenosine Diphosphate↗