Hello Mohammed I. Rushdi

Can you send me full protocol ? It will be more helpful for me. Thanks.

Article Dissection of Host Cell Signal Transduction during Acinetoba...

Method for lipid A extraction

A. baumannii ATCC 17978 lipid A was extracted using an ammonium hydroxide/isobutyric acid method [31] and subjected to negative ion matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Briefly, lyophilized cells (10 mg) were resuspended in 400 µl isobutyric acid/1M ammonium hydroxide (5∶3, v/v) and incubated in a screw-cap test tube at 100°C for 2 h, with occasional vortexing. Samples were cooled in ice water and centrifuged (2,000×g for 15 min). The supernatant was transferred to a new tube, diluted with an equal volume of water, and lyophilized. The sample was washed twice with 400 µl methanol and centrifuged (2,000×g for 15 min). The insoluble lipid A was solubilized in 100–200 µl chloroform/methanol/water (3∶1.5∶0.25, v/v/v). Analyses were performed on a Bruker Autoflex II MALDI-TOF mass spectrometer (Bruker Daltonics, Incorporated) in negative reflective mode with delayed extraction. Each spectrum was an average of 300 shots. A peptide calibration standard (Bruker Daltonics) was used to calibrate the MALDI-TOF. The ion-accelerating voltage was set at 20 kV. Dihydroxybenzoic acid (Sigma Chemical Co., St. Louis, MO) was used as a matrix. A few microliters of lipid A suspension (1 mg/ml) were desalted with a few grains of ion-exchange resin (Dowex 50W-X8; H+) in an Eppendorf tube. A 1 µl aliquot of the suspension (50–100 µl) was deposited on the target and covered with the same amount of the matrix suspended at 10 mg/ml in a 0.1 M solution of citric acid.

Mohammed I. Rushdi

Thank you so much.

I have another inquiry. Can you tell me some protocol about membrane separation from bacteria sample ? For your kind information, I want to separate outer membrane and Inner membrane structure of my Wild type and Mutant. I want to see what is main difference between this two. Because my phage can not infect my Mutant but WT can infect. Thanks again Mohammed I. Rushdi bhiya.