Qproteome Bacterial Protein Prep Kit

Protein isolation Bacteria - Mycobacterium smegmatis

Experiment
Protein isolation Bacteria - Mycobacterium smegmatis
Product
Qproteome Bacterial Protein Prep Kit from Qiagen
Manufacturer
Qiagen

Protocol tips

Upstream tips
- The liquid medium is aspirated from the Petri dishes. A proteinase inhibitor cocktail (Complete; Roche) is added to the Petri dishes and the dishes are frozen at −80°C.
Protocol tips
- Qproteome Bacterial Protein Prep kit (Qiagen) is added to each Petri dish. The whole content is transferred into a glass test tube and vortexed (10×30s cycles with 1 min breaks on ice). The beads and cell debris are removed by centrifugation (5 min at 5000g at 4°C) and are rewashed with lysis buffer.

- The proteins precipitation and resuspension protocol is the one provided by the publication.

Publication protocol

The liquid medium was aspirated from the Petri dishes, so that the biofilm pellicle dropped onto the glass beads. A proteinase inhibitor cocktail (Complete; Roche) was added to the Petri dishes, and the dishes were then frozen at −80 °C. Prior to protein isolation, the Qproteome Bacterial Protein Prep kit (Qiagen) was added to each Petri dish. The whole content of the Petri dish, including the glass beads, was transferred into a glass test tube and vortexed for 10× 30 s cycles with 1 min breaks on ice. The beads and cell debris were removed by centrifugation (5 min at 5000 g at 4 °C) and were rewashed with lysis buffer. The proteins were precipitated with 6 % TCA (w/v), incubated on ice for 1 h and centrifuged at 10 000 g at 4 °C for 15 min. The pellets were washed with ice-cold acetone and kept overnight in acetone at −20 °C. The samples were centrifuged (15 min at 10 000 g at 4 °C), and the protein pellets were resuspended in phosphate buffer and treated with a 2-D Clean-Up kit (GE Healthcare). The pellets were finally resuspended in sample buffer containing 7 M urea, 2 M thiourea, 4 % (w/v) CHAPS, 1 % pharmalyte 3–10, 65 mM DTT and bromophenol blue. The protein concentrations were measured using a 2-D Quant kit (GE Healthcare).

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Discussion

Discussion

2 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 - Mycobacterium smegmatis using Qproteome Bacterial Protein Prep Kit from Qiagen.

Paper title
Morphological and proteomic analysis of early stage air-liquid interface biofilm formation in Mycobacterium smegmatis.
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Manufacturer protocol

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

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