Qproteome Bacterial Protein Prep Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Colonies are aseptically scraped off culture plates and resuspended in 500 μL distilled water. - Bacterial cells are subsequently inactivated at 85°C for 30 minutes. - Inactivated cells are centrifuged at 8,000 rpm for 5 minutes and the supernatant discarded.	- Total bacterial protein was extracted following the manufacturer protocol. Instead of using 10mL of Native Lysis Buffer it is advised to use 1mL.

Upstream tips
- Colonies are aseptically scraped off culture plates and resuspended in 500 μL distilled water. - Bacterial cells are subsequently inactivated at 85°C for 30 minutes. - Inactivated cells are centrifuged at 8,000 rpm for 5 minutes and the supernatant discarded.
Protocol tips
- Total bacterial protein was extracted following the manufacturer protocol. Instead of using 10mL of Native Lysis Buffer it is advised to use 1mL.

Publication protocol

MTB colonies growing exponentially on Löwenstein–Jensen medium were aseptically scraped off culture plates and resuspended in 500 μL distilled water. Bacterial cells were subsequently inactivated at 85°C for 30 minutes. Inactivated cells were centrifuged at 8,000 rpm for 5 minutes and the supernatant discarded. The resultant pellets were collected and stored at –80°C. Total bacterial protein was extracted using a Qproteome bacterial protein prep kit. Briefly, the frozen bacterial pellets were thawed for 15 minutes on ice and resuspended in 1 mL native lysis buffer. Resuspensions were subsequently incubated on ice for a further 30 minutes. Next, the chilled suspensions were mixed two to three times by gentle swirling. The resultant lysates were centrifuged at 14,000× g for 30 minutes at 4°C, in order to collect the cellular debris. The cell-lysate supernatants containing the soluble fractions of the bacterial cell proteins were retained, and the concentration and purity of total proteins were determined using a nucleic acid/protein detector.

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Manufacturer protocol

Download the product protocol from Qiagen for Qproteome Bacterial Protein Prep Kit below.

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