CelLytic™ M

Protein isolation Bacteria - Staphylococcus aureus

Experiment
Protein isolation Bacteria - Staphylococcus aureus
Product
CelLytic™ M from Sigma-Aldrich
Manufacturer
Sigma-Aldrich

Protocol tips

Upstream tips
- Cells are harvested by centrifugation (5 min, 15,000 × g, 4 °C).
Protocol tips
- Cell pellets are resuspended in 850 μl of lysis buffer (90 mm Tris, pH 8.0, and 100 μm fluorocitrate), and bacteria are mechanically disrupted in a Fast Prep instrument at a speed of 6.0 for 30 s. Lysates are centrifuged (1 min at 15,000 × g and 4 °C).

Publication protocol

Bacteria were cultivated in LB-L as described before, and aliquots of the cultures (2 ml) were removed after 3, 6, 9, and 12 h of growth. Cells were harvested by centrifugation for 5 min at 15,000 × g and 4 °C, cell pellets were resuspended in 850 μl of lysis buffer (90 mm Tris, pH 8.0, and 100 μm fluorocitrate), and bacteria were mechanically disrupted in a Fast Prep instrument (Qbiogene, Heidelberg, Germany) at a speed of 6.0 for 30 s. Lysates were centrifuged for 1 min at 15,000 × g and 4 °C, and total protein concentrations of the supernatants were determined according to the method described by Bradford. Aconitase activities were assayed in cell-free supernatants as described previously. One unit of aconitase activity was defined as the amount of enzyme necessary for a ΔA240 min−1 of 0.0033. Citrate synthase activities were determined with the Citrate Synthase Assay Kit (Sigma) according to the manufacturer's recommendations, with the following modifications. Cell pellets were suspended in CelLytic M Cell Lysis Reagent, and cell-free supernatants were obtained as described above.

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Discussion

Discussion

5 years ago

Author: Hollie Fowler United Kingdom

How can I deal with my pellet being too viscous?

I am using 8M Urea to lyse my cells but after centrifugation my pellet is very viscous. This causes me trouble when I try to collect the supernatant. Is there any way to avoid it and do you think it will have an effect on the concentration of my protein?

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Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein isolation Bacteria - Staphylococcus aureus using CelLytic™ M from Sigma-Aldrich.

Paper title
Catabolite Control Protein E (CcpE) Is a LysR-type Transcriptional Regulator of Tricarboxylic Acid Cycle Activity in
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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for CelLytic™ M below.

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Videos

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