Protein isolation Bacteria - Escherichia coli

Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.

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6 hours ago

6 hours ago by Christos Karampelas Netherlands

Is a bacterial nuclease contamination possible during protein purification?

I am currently using a recombinant protein which shows metal-dependent DNase activity. Is it possible to pinpoint the source of the DNase activity after protein purification? More specifically, can I ensure that the DNase activity is not because of nuclease contamination from the E.coli that might have persisted and passed with the protein of interest during purification?

Discussion

2 years ago

2 years ago by 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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Found 4 matching solutions for this experiment

Upstream tips
- Capable of extracting bacterial microcompartments (BMCs).
Protocol tips
- The manufacturer protocol was followed.
Downstream tips
- Compatible with most downstream workflows such as electrophoresis, affinity purification, immunoprecipitation, protein interaction analysis, crosslinking and protein labeling.
Protocol tips
- Cells are lysed by sonication in lysis buffer [400 mM NaCl, 20 mM Tris⋅HCl, pH 8.0, lysozyme 600 μg/mL, 4 μL of Benzonase Dnase (Sigma), 0.5 mL of CelLytic B (Sigma), 1 tablet of EDTA-free protease inhibitor mixture (cOmplete; Roche), 1% (vol/vol) Triton X-100].
Upstream tips
- Capable of extracting bacterial microcompartments (BMCs).
Protocol tips
- The manufacturer protocol was followed.
Protocol tips
- The protocol was followed according to manufacturer’s instructions.
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