To demonstrate the procedure, electrospray-compatible solutions of myoglobin with neutral pH, in which binding activities are maintained for performing the ITEM-TWO experiments, are prepared. A stock solution was first prepared by dissolving 5.22 mg of myoglobin (lot # 60K7007, Sigma-Aldrich, Steinheim, Germany) in 1 mL of 200 mM ammonium acetate. Next, 50 µL of the stock solution was diluted with 450 µL of 200 mM ammonium acetate. The concentration of the resulting solution was determined to be 0.5 µg/µL using a QubitTM 2.0 Fluorometer (Carlsbad, California, USA). Finally, 100 µL of the 0.5 µg/µL myoglobin solution were further diluted to 200 µL with 200 mM ammonium acetate buffer, pH 7, to obtain a final concentration of 0.25 µg/µL. For each measurement, ca. 3 µL of the 0.25 µg/µL myoglobin solution were loaded into nanoESI capillaries using a microloader pipette tip (Eppendorf, Hamburg, Germany) and were electrosprayed directly.
Microloader pipette tip
Manufactured by Eppendorf
Sourced in Germany
The Microloader pipette tip is a specialized laboratory equipment designed for precise and accurate pipetting of small sample volumes. It features a narrow tip design that allows for the handling of micro-scale samples, making it suitable for applications that require meticulous liquid handling, such as DNA/RNA extraction, PCR setup, and other delicate procedures. The Microloader tip is compatible with a wide range of pipettes, ensuring versatility and flexibility in the laboratory.
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5 protocols using microloader pipette tip
1
Electrospray-compatible Myoglobin Preparation
2
Mass Spectrometric Analysis of Protein G
3
Purification and Characterization of RNAse S
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A stock solution of ca. 1 mg/mL was first prepared by dissolving the lyophilized powder (0.26 mg) of RNAse S (Lot # 52H7034, Sigma-Aldrich, Steinheim, Germany) in 0.26 mL of 200 mM ammonium acetate, pH 7.0. Then, 100 µL were transferred onto a Microcon centrifuge filter with a 3 kDa cutoff (Millipore Corp., Bedford, MA, USA) together with further 200 µL of 200 mM ammonium acetate solution. This solution was centrifuged for 30 min at 13,000 rpm and 23 °C. The eluate was discarded and 200 µL of 200 mM ammonium acetate solution were added onto the filter. This procedure was repeated for three times. Then, the filter was inverted, placed into a new tube, and centrifuged for 5 min at 4500 rpm at 23 °C. A resulting supernatant of approximately 800 µL was collected and the protein concentration was determined to be 0.78 µg/µL using a QubitTM 2.0 Fluorometer (Carlsbad, CA, USA) assay. For nano electrospray mass spectrometry 2.56 µL of the purified and concentrated RNAse S solution were diluted to a final concentration of 0.2 µg/µL with 7.44 µL of 10% methanol/200 mM ammonium acetate. For each measurement, ca. 3 µL of the RNAse S solution were loaded into nanoESI capillaries using a microloader pipette tip (Eppendorf, Hamburg, Germany).
4
Fluorescent Reporter Screening in Zebrafish Larvae
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Check all larvae under a fluorescence stereoscope for homogeneous fluorescent reporter expression, spontaneous inflammation and appropriate age-related development. Larvae are sorted in fresh E3 medium supplemented with 0.03% methylene blue and MS-222 (1X; 160 mg/L). Larva sorting is preferred at about 3 to 4 dpf. If necessary, orient larvae in a lateral position using a flexible Eppendorf Microloader Pipette Tip for better visualization of fluorescent reporter expression. Place larvae in transparent 250 mL containers filled with 50 mL E3 medium supplemented with methylene blue until 6 dpf. Close the containers with holed lids and incubate them at 28°C.
5
Lateral Imaging of Larval Gut
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Larvae are mounted lateral side down in 1% low gelling temperature agarose dissolved in PBS, over glass bottom culture dishes and overlaid with PBS for imaging. Use a flexible Eppendorf Microloader Pipette Tip to orient larvae in a lateral position for better visualization of fluorescent reporter expression. Z-stack acquisition is performed with Zeiss Zen 3.0 (blue edition) software for multipositions in an automated and motorized inverted microscope (Zeiss Axio Observer) using a 10× objective (NA 0.3) and a mercury lamp. Position the larvae to have the more distal part of the intestine centered anterior posteriorly and dorsal ventrally in the image. Set the lumen of the gut as the z-center level. Image each larva in the channels brightfield and mCherry (ex: 585 nm, em: 610 nm band pass filters) in 49 focal planes (149 μm range; 3 μm z step) so that all the gut depth is visible.
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