Use Of Lysis Buffer Research Articles

Lysing Yeast Cells with Glass Beads for Immunoprecipitation.

Yeast cells display cell walls that must first be broken before the addition of detergents for lysis. This method describes the use of glass beads in combination with a mechanical bead beater to disrupt cell walls of both Saccharomyces cerevisiae or Schizosaccharomyces pombe directly in a nonionic detergent Lysis buffer containing 0.1% Nonidet P-40. Alternatively, this protocol can be applied for the lysis of yeast cells in Lysis buffer without detergent; upon completion of the bead beating, Triton X-100 is added to complete lysis. Yeast cells are cultured and collected while in log phase before being washed once and mixed together with glass beads in a tube. The applied shaking process facilitates disruption of the cell walls, upon which separation of yeast and glass beads is accomplished by forcing lysed cells through a hole created in the bottom of the tube during the centrifugation process. An alternative bead-beating protocol details the use of Lysis Buffer 2, which does not contain detergents and calls for the addition of Triton X-100 after cell lysis in the presence of glass beads. Use of Lysis Buffer 2 without detergent may avoid bubble and foam formation during the bead-beating process that could potentially denature proteins.

Viral RNA Degradation Makes Urine a Challenging Specimen for Detection of Japanese Encephalitis Virus in Patients With Suspected CNS Infection.

BackgroundJapanese encephalitis virus (JEV) is a leading cause of central nervous system (CNS) infections in Asia and results in significant morbidity and mortality. JEV RNA is rarely detected in serum or cerebrospinal fluid (CSF), and diagnosis of JEV infection is usually based on serological tests that are frequently difficult to interpret. Unlike serum or CSF, urine is relatively easy to obtain, but, to date, there has been minimal work on the feasibility of testing urine for JEV RNA.MethodsWe investigated the use of lysis buffer and a Microsep device to optimize urine storage for detection of JEV RNA by reverse transcription real-time polymerase chain reaction (RT-qPCR). The best of the studied methods was then evaluated in consecutive patients admitted to the hospital with suspected CNS infections in Laos.ResultsWe demonstrated degradation of JEV RNA in urine after even short storage periods at 4°C or –80°C. Although there was no advantage in using a Microsep concentration device alone, immediate addition of lysis buffer to fresh urine improved the detection of JEV RNA at the limit of detection.ConclusionsIn 2 studies of 41 patients with acute encephalitis syndrome, 11 (27%) were positive for JEV IgM in CSF and/or serum, and 2 (4.9%) were JEV RT-qPCR positive from throat swabs. JEV RNA was not detected in any of these patients’ urine samples. However, lysis buffer was only used during a prospective study, that is, for only 17/41 (41%) patient urine samples. Our findings suggest a need for larger studies testing urine for JEV RNA, with urine collected at different times from symptom onset, and using lysis buffer, which stabilizes RNA, for storage.

Improving recovery and stability of touch DNA

Touch samples by their nature do not contain large amounts of biological material, especially compared to biological fluids such as blood, semen or saliva. Consequently, more careful recovery and storage are desirable in order to maximise the amount of DNA available. The main aim of the work presented is to determine whether detergent-based wetting agents increased DNA yield from touch samples when compared to water as a wetting agent. The results show that the use of the detergent-based lysis buffer led to greater DNA recovery from the fingerprints than when distilled water was used. The use of the lysis buffer in the touch DNA sample collection improved DNA recovery and stability over 24h post-collection.

Quantification of fatty acids in erythrocytes and plasma by fast gas chromatography.

As a result of the heterogeneous nature of lipid classes in complex biological matrices such as plasma and erythrocytes, it is imperative to have a robust and validated methodology for fatty acid quantification. The effective method presented here combines available methodology of fast gas chromatography and an improvement of the sample preparation methodology before injection into the gas chromatograph. This methodology ensures complete transesterification and quantification of total and individual fatty acids (and not only in relative amounts) by addition of internal standards. We considered sample preparation key, and we established the use of lysis buffer and ethanol for erythrocytes and plasma sample preparation, respectively. Fatty acid profile was determined by acid methylation and fast gas chromatography equipped with a flame ionization detector. The triacylglycerol 13:0, phosphatidylcholine 23:0, and methyl esters 21:0 were used as internal standards. Within the linearity of the calibration, the ratio of the peak area of each fatty acid over the peak area of the internal standard was constant (coefficient of variation≤2.5). Satisfactory repeatability <15% and intermediate reproducibility <15% were observed. Finally, this validated method was applied to a pre-clinical trial that investigated the impact of dietary fats on accretion of specific fatty acids in plasma and erythrocytes.

Proteome Approach as a Tool for the Efficient Separation of Seed Storage Proteins from Buckwheat

Two-dimensional electrophoresis (2-DE) was executed to separate the seed storage proteins from the buckwheat. The proteins extracted from the whole seed proteins were better separated and observed in the use of lysis buffer. Using this method, the highly reproducible isoelectric focusing (IEF) can be obtained from polyacrylamide gels, and IEF from the polyacrylamide gel at all the possible pH range (5.0-8.0) was more easily separated than IPG (immobilized pH gradient) gels. The polyacrylamide gels in the first dimension in 2-DE was used to separate and identify a number of whole seed proteins in the proteome analysis. In this new apparatus using 2-DE, 27cm in length of plate coated with polyacrylamide gel was used and the experiment was further investigated under the various conditions.

Comparison of three methods for concentration of rotavirus from artificially spiked shellfish samples

Materials and Methods Shellfish is cheap and highly proteinaceous food whose consumption has increased globally. Shellfish by virtue of their filter feeding habit can concentrate pathogenic organisms such as enteric viruses and bacteria present in contaminated water [1]. A number of human enteric viruses including norovirus, aichi, rotavirus, enterovirus, adenovirus, astrovirus, sapovirus, Hepatitis A and Hepatitis E virus have been detected in shellfish, although not all have been clearly linked with documented disease outbreaks [2, 3]. Faecal indicator bacteria routinely used to access sanitary quality of water is successful only in prevention of shellfish borne infection of bacterial origin and have limited predictive value for pathogenic viruses [4]. Hence there is need for methods that can be used for direct virological examination of samples in order to access the health risk due to enteric viruses. Identification of enteric virus from shellfish is difficult due to the low concentration of virus and the presence of substances liable to interfere with enzyme system used for amplification [5,6]. The main inhibitors in shellfish are acid polysaccharides [7]. Effective preliminary sample treatment steps such as concentration of virus from shellfish are therefore essential before RNA/DNA isolation for final PCR accuracy and reproducibility [8]. The aim of the study is to compare three methods namely Proteinase K treatment [9, 10], precipitation with polyethylene glycol 8000 [11], and use of lysis buffer [12] for concentration of rotavirus from shellfish after artificially spiking the shellfish with known concentration of faecal sample containing rotavirus.

MiR‐TRAP: A Benchtop Chemical Biology Strategy to Identify microRNA Targets

miR‐TRAP: A Benchtop Chemical Biology Strategy to Identify microRNA Targets

Bufferless lysis of erythrocytes for isolation of hemoglobin using modified cellulose acetate membranes

This paper presents a modified cellulose acetate membrane prepared using a dry casting technique that can be used to perform lysis of erythrocytes and isolation of hemoglobin. Isolation of hemoglobin is thus achieved without the use of lysis buffers. Cellulose acetate (CA) membranes are embedded with ammonium chloride (NH4Cl) and potassium bicarbonate (KHCO3), which act as lysing agents. The presence of embedded salts is confirmed using EDS analysis. The pores in the CA membrane act as filters. The average pore size in these membranes is designed to be 1.5 μM, as characterized by SEM analysis, so that they allow hemoglobin to pass through and block all other cells and unlysed erythrocytes present in blood. When a drop of blood is added to the membrane, the NH4Cl and KHCO3 embedded in the membrane dissolve in plasma and lyse the erythrocytes. The filtered hemoglobin is characterized using UV-Vis Spectroscopy. The results indicate extraction of higher concentration of hemoglobin compared with conventional methods.

Quantitative detection of circulating endothelial cells in vasculitis: comparison of flow cytometry and immunomagnetic bead extraction.

Circulating endothelial cells (CECs) are biomarkers for endothelial cell (EC) injury and are quantified using immunomagnetic bead extraction (IBE), or flow cytometry (FC). Reports suggest that there is good agreement between these methods for CEC quantification. We examined levels of agreement between these techniques in children with systemic vasculitis. We added HUVEC or human pulmonary artery EC to whole blood to optimize FC gating strategies for EC. EC-optimized FC was then compared with IBE for CEC enumeration in 25 children with vasculitis and 20 healthy controls. Using Bland-Altman analysis, agreement between IBE and EC-optimized FC was poor in children with vasculitis (n = 25) and healthy controls (n = 20): IBE consistently detected higher values than the EC-optimized FC method: the mean difference between the two techniques was 60 CECs mL(-1), 95% CI +/-374 CECs mL(-1) (paired analyses of 45 individuals). Agreement was poorest for vasculitis patients: mean difference (IBE - EC-optimized FC) 120 CECs mL(-1), 95% CI +/-460 CECs mL(-1) (P = 0.018). We identified three reasons for this discrepancy: (i) sub-optimal FC gating parameters previously used for detecting CECs; (ii) inherent lack of sensitivity of FC compared with IBE for CEC rare event detection; and (iii) use of lysis buffers required for FC causing CEC lysis. There was poor agreement between EC-optimized FC and IBE for the quantification of CECs from children with active vasculitis and controls. We emphasize that in this clinical setting the two techniques are not directly comparable when comparing results obtained using these different methodologies.

Comparison of cell disruption methods for determining β-galactosidase activity expressed in animal cells

To determine β-galactosidase activity expressed in animal cells, the cells are often disrupted. In this study, four different cell disruption methods (freezing and thawing, sonication, homogenization, and lysis buffer) were compared with regard to their efficiency in the determination of β-galactosidase activity. Further, the cell lines' susceptibility to cell disruption methods was investigated by employing three different animal cell lines (HeLa HH, Vero, and HeLa S3 cells) infected by recombinant vaccinia virus (vSC8) expressing β-galactosidase gene. Regardless of cell lines used, three different cell disruption methods except homogenization did not show any significant difference in the final β-galactosidase activity recovered from the cells. Homogenization was inefficient, and required up to 300 strokes to recover β-galactosidase activity fully from HeLa S3 cells. The use of lysis buffer was recommendable because of its convenience. However, if there was no need for an immediate assay, it was convenient to keep cell samples collected during the culture frozen at −20°C until the assay. Since thawing of the cells was enough to recover the β-galactosidase, further treatment on the frozen cells after thawing was unnecessary.