1 | PEB (4x) | protein extraction buffer

background | PEB is an extraction buffer for disruption and solubilisation of total protein from plant tissue and algal cells. The use of the anionic detergent LDS together with the recommended procedure (combination of sonication and freeze/thaw cycles) has been shown to increase the quantity of solubilised and non-degraded proteins when compared to other methods of cell disruption (see reference). The estimated hands-on time for the recommended procedure is 20-30 minutes for 1-2 samples. Expected yields will be 1.5-6.0 µg/µl total protein (recovered from standard procedure) depending on the starting material, e.g. its biological stage, homogenization method used (bead beater vs. sonication).

quantity | 5x2 ml (4x stock); allows up to 75 isolationsof plant material (using 500 µl 1x PEB for 100 mg fresh weight) or 190 isolations of algal material(using 200 µl 1x PEB for cell amounts corresponding to 4-10 ug total chlorophyll)

specificity | PEB has been tested on a wide range of species and tissues from higher plants, mosses, lichens, algae, diatoms, dinoflagellates, and cyanobacteria. Extracts may be quantified using detergent (LDS) compatible methods, and have been shown to give highly reproducible and quantitative results in subsequent SDS PAGE gel electrophoresis, Western Blotting, and immunoprecipitation. Most of Agrisera commercial antibodies are tested on plant or algal samples extracted with this buffer. An example can be found here.

storage | 1 year in 4°C

reference | Brouwer et al. (2011) The Impact of Light Intensity on Shade-Induced Leaf Senescence. Plant Cell Environ. Dec. 15 (ahead of print).

Kosawang et al. (2011) Hydrogen yield from a hydrogenase in Frankia R43 at different levels of the carbon source propionate. Journal of Environmental Management, Jan 26

Rapid protein isolation from multiple samples can be successfully done using:the FastPrep®-24 Instrument (MP) or Precellys® (Bertin Technologies). For isolation of protein with microalgae using FastPrep®-24, 3 rounds of 60s @ 6.5 m/s were applied or 3 times 20 sec at 6,500 rpm with 5 sec pauses using Precellys.

For help with protein extraction from algal and cyanobacterial cells you are welcome to contact us.

Agrisera tips for a successful protein extraction from plant and algal samples

• use extraction method which provides good yields and consistent results. Agrisera has recently validated Precellys® device from Bertin Techonologies, as fullfilling such criteria

• use a protease inhibitor always made from a fresh stock

• keeping a sample volume in a range of 0.2-0.5 ml will contribute to better extraction results (per 100 mg of plant tissue or 4-10 ug of chlorophyll for algal samples

• keep material cool at all times

• for photosynthetic tissues/algal samples intense green pellet indicates that extraction is not optimal and needs to be repeated

• protein extracts should be stored at -80°C aliquoted and used only ONCE

• reducing agent should be added before heating just before loading on a gel

• Agrisera protein extraction buffer - especially prepared for isolation of plant and algal proteins can be used

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