Biomedical subjects

Alamgir Khan

Publications and source records attributed to Alamgir Khan.

4 recordsLinked to original sources

PubMed2006

Simple urinary sample preparation for proteomic analysis.

Since the completion of the human genome sequence, attention has now focused on establishing reference maps of body fluids such as plasma and urine for detecting diagnostic markers of disease. Although some progress has been made, challenges still remain in the development of an optimal sample preparation method for proteomic analysis of urine. We have developed a simple and efficient urine preparation method for two-dimensional (2-D) gel electrophoresis which involves precipitation of proteins with simultaneous desalting. Acetonitrile precipitation produced 2-D gel separations with the highest resolution and the greatest number of protein spots compared to precipitation by other organic solvents. The method was applied to observe changes in the urinary proteome over a 6 week period and to establish a reference map of a healthy subject. A total of 339 proteins from 159 genes was identified from healthy male urine by peptide mass fingerprinting. The profiles of the urinary proteome at three times in 1 day and on four different days were compared and were found to vary in number and spatial location of the proteins on the map. The method was also shown to be applicable to the higher concentrations of protein found in the urine of an ovarian cancer subject. We have developed a facile and robust method for preparing urine for 2-D gels that will encourage further use of urine.

Adult↗

PubMed2005

New urinary EPO drug testing method using two-dimensional gel electrophoresis.

UNLABELLED: We present a two-dimensional electrophoresis (2DE) method for the detection of the drug, recombinant erythropoietin (rHuEPO) in urine and its separation from endogenous erythropoietin (HuEPO). This method involves a one-step acetonitrile precipitation of the proteins in urine, addition of an internal standard, two-dimensional gel electrophoresis (2D PAGE), a single Western blot and chemiluminescent immunodetection. RESULTS: The 2DE method separates HuEPO and rHuEPO isoforms by both iso-electric point and molecular mass. We have identified several urinary proteins with which the monoclonal EPO antibody used in the current test has non-specific binding. The iso-electric points of these cross-reactive proteins overlap with HuEPO and rHuEPO however, they separate distinctly by the 2DE method. Alpha-2-HS-glycoprotein (HSGP) was identified by peptide mass fingerprinting as one of the urinary cross-reacting proteins, and commercially available purified HSGP was chosen to be added into urine samples as an internal standard prior to separation. Software (EpIQ) was specifically developed that applies four separate criteria to the detection of the migration of rHuEPO and HuEPO relative to the internal standard. CONCLUSION: The combination of sample preparation, two-dimensional separation, internal standard, standardized blotting procedures and image analysis software enables the 2DE test for rHuEPO in urine to be performed reproducibly and accurately.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2003

Testing the nematophagous biological control strain Paecilomyces lilacinus 251 for paecilotoxin production.

Paecilomyces lilacinus is a nematophagous fungus currently developed as a biological control agent. In order to evaluate potential toxin production, culture extract and concentrated culture supernatant of P. lilacinus strain 251 were tested against Gram-negative and Gram-positive bacteria. High-performance liquid chromatography analysis was carried out to compare the chromatograms of P. lilacinus strain 251 with the chromatogram of known paecilotoxin. It was found that the 251 strain of P. lilacinus did not produce detectable levels of paecilotoxin or other toxins with antimicrobial activity.

Animals↗

PubMed2003

Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels.

The filamentous fungus Paecilomyces lilacinus is currently developed as a biocontrol agent against plant parasitic nematodes. Nematode eggs and cuticles are the infection sites for biocontrol agents that penetrate by the production of lytic enzymes. P. lilacinus was cultured in liquid media and proteases and chitinases were induced by the introduction of egg yolk and chitin, respectively. A serine protease was purified from a culture medium using Sepharose-bacitracin affinity column. The protease occurred in three forms, two of which were C-terminally truncated. Chitinase activity was also observed in the culture supernatant, and after separation by isoelectric focusing six proteins were detected that showed activity. Chitinase activity was further confirmed on non-denaturing one-dimensional (1D) and two-dimensional (2D) gels using a sandwich assay with glycol chitin as a substrate. Two of the proteins had similarities with endochitinases as shown by their N-terminal amino acid sequences.

Amino Acid Sequence↗