Protein isolation Bacteria - Borrelia burgdorferi

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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4 years ago

4 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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Upstream tips
- There is a previous cell lysate step.
Protocol tips
- Total cell lysate is subjected to ultracentrifugation (435,700 × g, 1 h, 4°C) to pellet membrane vesicles.

- Membrane pellets are rinsed in 1.0 ml of HN buffer with protease inhibitors, reisolated by ultracentrifugation, and suspended in 200 to 400 μl of HN buffer plus complete protease inhibitor cocktail with the aid of a coated homogenizer.
Downstream tips
- Protein precipitation is performed following Trichloroacetic Acid Solution, MP Biomedicals™ protocol (Fisher Scientific).
Upstream tips
- Previous isolation of membrane proteins with Gyros IncSupplier Diversity Partner REXXIP HN BUFFER 25 ML PER VI DFS Item (Fisher Scientific).
Protocol tips
- Membrane proteins are precipitated by the addition of 8 volumes acetone (Fisher), 1 volume of a saturated trichloroacetic acid solution (Fisher). Incubation: −20°C for 45 min. After, are subjected to centrifugation (16,000 × g, 20 min, 4°C), rinsed in 80% cold acetone in distilled water, air dried, and solubilized.
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