Automated Protein Quantification with the Biomek-FX Liquid Handler System v2

This protocol details steps to perform the protein quantification (Lowry-based) assay by using a Biomek FX liquid handler system. It is optimized to assay a full 96-well plate of protein samples in duplicate with a separate (control) plate for BSA standards. You will need a plate reader to measure the samples and standards. This protocol works best as part of a full proteomic sample preparation workflow with: Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System and Automated Protein Normalization and Tryptic Digestion on a Biomek-FX Liquid Handler System

Estimating Microbial Population Data from Optical Density

The spectrophotometer has been used for decades to measure the density of bacterial populations using the turbidity expressed as optical density – OD. However, the OD alone is an unreliable metric and is only proportionately accurate to cell titers to about an OD of 0.6. The relationship between OD and cell titer depends on the configuration of the spectrophotometer, the length of the light path through the culture, the size of the bacterial cells, and the cell culture density. We demonstrate the importance of plate reader calibration to identify the exact relationship between OD and cells/ml. We use four bacterial genera and two sizes of micro-titer plates (96-well and 384-well) and show that the cell/ml per unit OD depends heavily on the bacterial cell size and plate size. We applied our calibration curve to real growth curve data and conclude the cells/ml – rather than OD – is a metric that can be used to directly compare results across experiments, labs, instruments, and species.

Pembentukan Biofilm Pseudomonas aeruginosa pada Beberapa Media Cair

Pseudomonas aeruginosa merupakan bakteri Gram negatif berbentuk batang bersifat patogen oportunistik yang menjadi penyebab utama infeksi nosokomial dan mampu membentuk biofilm pada media pertumbuhan, biofilm sering mengakibatkan pengobatan penyakit infeksi menjadi lebih sulit. Media pertumbuhan bakteri ada beberapa jenis, komposisi dan merek. Tujuan penelitian ini adalah untuk mengetahui kemampuan P. aeruginosa dalam membentuk biofilm pada beberapa media biakan cair. P. aeruginosa diisolasi dari sampel klinis dari rumah sakit, media cair yang digunakan adalah nutrien broth, laktosa broth, brain-heart infusion (BHI), luria bertani broth, dan tripticase soy broth. Uji pembentukan biofilm menggunakan metode microtiter plate culture technique, kemampuan pembetukan biofilm diukur berdasarakan optical density dengan menggunakan microtiter plate reader pada panjang gelombang 570nm, dengan pewarnaan crystal violet 0,1%, setelah inkubasi 24 jam pada suhu 37oC, dengan replikat 8 kali. Hasil penelitian menunjukkan bahwa P. aeruginosa memiliki kemampuan membentuk biofilm pada nutrient broth 0,926±0,081, laktosa broth 0,521±0,041, BHI 1,283±0,031, luria bertani 1,301±0,043, dan media trypticase soy broth 1,563±0,032. Pembentukan biofilm tertinggi pada trypticase soy broth, dan terendah pada laktosa broth, sedangkan pada media BHI dan luria bertani kemampuan pembentukan biofilm yang setara. Kesimpulan penelitian ini adalah P. aeruginosa memiliki kemampuan yang berbeda dalam membentuk biofilm ketika ditumbuhkan pada media cair yang berbeda.Kata kunci : Biofilm, Pseudomonas aeruginosa, media cair

FPCount protocol - Full protocol v1

FPCount is a complete protocol for fluorescent protein calibration, consisting of: 1. FP expression/purification using Thermo's HisPur Cobalt Resin. 2. FP concentration determination in a microplate reader. 3. FP fluorescence quantification in a microplate reader. Results can be analysed with the corresponding R package, FPCountR. --- Summary 1. Expression 2. Harvesting/Washing 3. Lysis 4. Fractionation 5. Gel1: Verification of Expression/Fractions 6. Purification 7. Gel2: Verification of Purification 8. Protein concentration and buffer exchange 9. Quantification of FP concentration (part1) 10. Quantification of FP fluorescence 11. Quantification of FP concentration (part2) 12. Protein Storage 13. Calibration of Plate Reader

Microplate-based DNA Quantification with EzFluoroStain DNA reagent v1

This protocol offers an safer alternative to the ethidium bromide-based DNA quantification protocol, utilizing an DNA-selective dye EzFluoroStain DNA (WSE-7130, ATTO corporation, Tokyo, Japan). This protocol allows to quantify about 2-1000 ng DNA/well, using a standard fluorescent plate reader.

FPCount protocol - in-lysate (purification free) protocol v1

FPCount is a complete protocol for fluorescent protein calibration, consisting of: 1. FP expression and production of cell lysates. 2. FP concentration determination in a microplate reader. 3. FP fluorescence quantification in a microplate reader. Results can be analysed with the corresponding R package, FPCountR. This in-lysate version of the protocol uses the ECmax protein quantification protocol of FPs in lysates and does not require His-tag purification of the FPs. Note that it is only suitable for FPs with entries in FPbase. If you want to verify or validate results, it's recommended you follow the 'short' protocol, which requires FP purification, or the 'complete' protocol, which requires FP purification and compares three protein quantification methods. --- Summary 1. Expression 2. Harvesting/Washing 3. Lysis 4. Fractionation 8. Protein concentration and buffer exchange 9. Quantification of FP concentration (part1) 10. Quantification of FP fluorescence 12. Protein storage 13. Calibration of Plate Reader

FPCount protocol - Short protocol v1

FPCount is a complete protocol for fluorescent protein calibration, consisting of: 1. FP expression/purification using Thermo's HisPur Cobalt Resin. 2. FP concentration determination in a microplate reader. 3. FP fluorescence quantification in a microplate reader. Results can be analysed with the corresponding R package, FPCountR. This short version uses the ECmax protein quantification protocol, and is only suitable for FPs with entries in FPbase. If you want to verify or validate results, it's recommended you follow the complete protocol, which describes three protein quantification methods. The short protocol also skips the SDS-PAGE steps. If you require these, please see the complete protocol. --- Summary 1. Expression 2. Harvesting/Washing 3. Lysis 4. Fractionation 6. Purification 8. Protein concentration and buffer exchange 9. Quantification of FP concentration (part1) 10. Quantification of FP fluorescence 12. Protein storage 13. Calibration of Plate Reader