Protocol using Hielscher Multi-Sample Sonicators in Biofilm and Proteomic Analysis

The use of multi-sample sonicators, such as the VialTweeter Multi-Tube Sonicator and the UIP400MTP Microplate Sonicator, streamlines laboratory workflows for high-throughput applications. Both devices allow precise and uniform ultrasonic processing across multiple samples, minimizing variability and enhancing reproducibility. This is particularly advantageous in experiments requiring consistent sample lysis, biofilm detachment, protein extraction or proteolytic digestion, where manual or single-sample methods are less efficient and prone to inconsistency.

Streamline Workflows with Multi-Sample Sonicators

The UIP400MTP, for instance, enables simultaneous ultrasonic treatment of microplate wells, ensuring uniform detachment of biofilms or cellular material. Meanwhile, the VialTweeter offers precise lysis and homogenization of multiple tube samples without cross-contamination. These sonicators significantly reduce processing times, improve experimental reproducibility, and maintain high sample integrity, making them indispensable tools in microbiology, proteomics, and molecular biology research.
Below is a detailed protocol exemplifying the use of the Hielscher multi-sample sonicator models, the VialTweeter Multi-Tube Sonicator and the Microplate Sonicator UIP400MTP, in a study involving E. coli biofilm formation and subsequent proteomic analysis.

Protocol: Biofilm Formation, Detachment, and Proteomic Analysis Using Hielscher Multi-Sample Sonicators

1. Bacterial Culture Preparation

• Culture E. coli bacteria (strain W3110) at 30°C, a temperature favorable for E. coli biofilm formation, in tryptone broth (TB) medium containing:
  – 10 g/L tryptone
  – 5 g/L NaCl
• Supplement with antibiotics as needed to ensure plasmid retention.
• For microscopy and promoter activity studies, use the W3110 E. coli strain with a genomic enhanced GFP (eGFP) reporter under the control of the rplL promoter.
• Utilize GFP reporter plasmids for various promoters (e.g., csgA, csgD, fliA, fliC, flhD ) to measure promoter activities.

2. Biofilm Cultivation and Detachment

• Seed 1.5 mL of diluted bacterial culture per well into a 12-well plate (CellStar 12-well plates, Greiner Bio-One).
• Incubate to allow biofilm formation.
• Remove the planktonic (non-adherent) culture carefully.

– Wash each well with 500 µL PBS.
– Detach attached cells by sonicating the 12-well plate in the UIP400MTP microplate sonicator. The UIP400MTP ensures a uniform detachment by delivering consistent ultrasonic energy across all wells.
– Sonication settings: 60% amplitude, cycle mode: 60 sec ON / 30 sec OFF.

3. Cell Harvesting and Lysis

• Centrifuge detached biofilm cells at 4,500 g for 5 minutes.
• Wash pelleted cells with PBS.
• Resuspend cells in 100 µL of 2% sodium lauroyl sarcosinate (SLS) and incubate at 95°C for 15 minutes.
• Sonicate using the VialTweeter Multi-Tube Sonicator to ensure complete cell lysis.
  – Sonication settings: 100% amplitude, 50 s ON / 10 s OFF for 10 min total run time; keep samples on ice.

4. Reduction and Alkylation

• Add 5 mM Tris(2-carboxyethyl)phosphine (TCEP) to reduce disulfide bonds and incubate at 95°C for 15 minutes.
• Alkylate proteins with 10 mM iodoacetamide for 30 minutes at 25°C.

5. Protein Quantification and Digestion

• Centrifuge lysates to clear debris.
• Quantify total protein content using the Pierce BCA Protein Assay Kit.
• Digest 50 µg total protein with 1 µg trypsin in a solution containing 2% SLS and 100 mM ammonium bicarbonate.
• Incubate digestion reaction overnight at 30°C.

6. Peptide Purification

• Precipitate SLS by adding 1.5% trifluoroacetic acid (TFA), then centrifuge.
• Purify peptides using C18 microspin columns.
• Dry purified peptides and resuspend in 0.1% TFA.

7. Liquid Chromatography-Mass Spectrometry (LC-MS/MS)

• Analyze peptides on a Q-Exactive Plus mass spectrometer coupled with an Ultimate 3000 RSLC nano system.
• Separate peptides using a reverse-phase HPLC column (75 µm × 42 cm) packed with C18 resin (2.4 µm).
• Apply a gradient from 2% to 35% solvent B (acetonitrile with 0.15% formic acid) over 140 minutes.
• Acquire data using high-resolution MS scans followed by tandem MS/MS scans of the 10 most intense ions.

8. Data Analysis

• Process raw LC-MS data using Progenesis software.
• Export .mgf files and analyze them with MASCOT.
• Perform peptide quantification using SafeQuant for quality control and false-discovery adjustment.
• Conduct all experiments in duplicates or triplicates to ensure reproducibility.

Multi-Sample Sonicators for High-throughput Sample Preparation

The multi-sample sonicator models VialTweeter and UIP400MTP significantly enhance the precision and efficiency of experimental workflows, especially in biofilm research and proteomics. Their ability to process multiple samples uniformly ensures high-throughput capacity without compromising data quality. These benefits, combined with the streamlined workflows outlined above, make Hielscher multi-sample sonicators essential tools for modern biological research.

Design, Manufacturing and Consulting – Quality Made in Germany

Hielscher ultrasonicators are well-known for their highest quality and design standards. Robustness and easy operation allow the smooth integration of our ultrasonicators into industrial facilities. Rough conditions and demanding environments are easily handled by Hielscher ultrasonicators.

Hielscher Ultrasonics is an ISO certified company and put special emphasis on high-performance ultrasonicators featuring state-of-the-art technology and user-friendliness. Of course, Hielscher ultrasonicators are CE compliant and meet the requirements of UL, CSA and RoHs.