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1 | PEB (4x) | protein extraction buffer

97 €

AS08 300  |  extraction buffer for quantitative isolation of total soluble/membrane protein from plant tissue and algal/bacterial cells, optimized for subsequent western blot detection in denatured conditions

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AS08 300

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product information
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 number 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 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).

Host
Clonality
Clone
Purity
Format
Quantity 5 x 2 ml (4x stock) allows up to 75 isolations of plant material (using 500 ĩl 1x PEB for 100 mg fresh weight) or 190 isolations of algal material (using 200 ĩl 1x PEB for cell amounts corresponding to 4-10 ĩg total chlorophyll)
Reconstitution
Storage

stable at RT for at least 1 month; short term storage (6 month) at 4°C and long term storage (1 year or more) at -20°C

Tested applications

protein extraction

Related products
Additional information

Buffer components (4x): contains ~ 40% v/v glycerol [HOCH2CH(OH)CH2OH], Tris-HCl [NH2C(CH2OH)3 · HCl] pH 8.5, LDS [CH3(CH2)11OSO3Li], EDTA [(HO2CCH2)2NCH2CH2N(CH2CO2H)2]

It is recommended to include a protease inhibitor (not supplied with this buffer) from a freshly made stock while preparing the ready-to-use 1x PSB.

PEB has been optimized for quantitative small-scale preparation of whole protein extracts from plant/algal tissue. Extraction using the procedure described below will result in maximum yield of proteins and diminish protein degradation and aggregation.

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.

PEB has been tested on a wide range of species and tissues from higher plants, mosses, lichens, algae, diatoms, dinoflagellates, and cyanobacteria.

application information
Recommended dilution
Expected | apparent MW
Confirmed reactivity 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.
Predicted reactivity
Not reactive in
Additional information
Selected references 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


Before you start
Prepare sufficient 1x PEB for all samples by diluting 4x stock (the pH of your 1x PEB should be between 8.25 and 8.75). It is recommended to include a protease inhibitor (not supplied with this buffer) from a freshly made stock while preparing the ready-to-use 1x PEB to increase the yield of non-degraded protein in the extract. We recommend including 1:50 vol/vol from a freshly prepared 50x stock (in 1x PEB) to give the desired final concentration recommended by the manufacturer (e.g. 0.1 mg/ml for Pefabloc SC, Roche).

The total volume of 1x PEB required is dependent on the sample type and amount of tissue used: for 100 mg fresh plant tissue we recommend 500 µl 1x PEB; for algal samples (corresponding to 4-10 µg total chlorophyll) we recommend 200 µl 1x PEB. Keeping sample volumes in a range of 0.2-0.5 ml has been found to contribute to better extraction results, an upscale in volume is not recommended.

Material preparation

plant tissue:
weigh and snap freeze in liquid nitrogen and store at -80°C until processing.
algal cultures: centrifuge to form a pellet or collect on filters (e.g.GF/F or polycarbonate) and freeze at -80°C until processing.



 
Extraction

1

grind frozen material in liquid N2 in a pre-chilled mortar with a pestle to a fine powder and transfer to a 1.5 ml tube

 

keep material cool at any time during grinding, avoid spillage

 

2

add 1x PEB and immediately freeze sample in liquid N2

 

500 µl for 100 mg plant tissue or 200 µl for cells corresponding to 4-10 µg total chlorophyll; keep tube upright to hold sample at the bottom of the tube

 

3

carefully subject sample to sonication just until sample is thawn, re-freeze sample immediately in liquid N2 to avoid heating

 

optimal results will be obtained using a microtip sonicator (e.g. Branson Ultrasonics Model 450)at low settings of about 30%; waterbath sonicators may also be used though this may lead to slightly less reproducible protein recovery rates;

 

4

repeat sonication step (3) depending on species, place on ice until all samples are processed

 

for higher plants 2-3 cycles, for cyanobacteria 3 cycles, for Chlamydomonas 2 cycles, for Heterosigma, Thalassiosira and Trichodesmium 1 cycle

 

5

centrifuge your samples for 3 min at 10 000 x g to remove insoluble material and unbroken cells, the pellet should be white/light-grey

 

an intense green color of the pellet indicates that disruption was not optimal and extraction conditions need to be adjusted (e,g, improved grinding and/or repeated sonication steps) using a new sample

 

6

transfer supernatant to new tube using a pipette, be careful not carry over debris

 

expect ~400 µl supernatant for the plant and ~150 µl for cyanobacterial/algal samples; collecting supernatant with a pipette as 2 x 200 (or 2 x 75 µl) reduces the risk of disturbing the pelleted debris

 

 

 

 

 

Protein determination
Assay total protein content of recovered supernatant using a detergent compatible assay. Based on the amount and/or tissue of the species used you may expect a protein content of 1.5-6 µg/µl.


Storage
Protein extracts may be stored for 24 hrs at +4°C or up to 6/12 month at -20°C/-80°C. We recommend to aliquot samples. Re-freezing protein samples may induce degradation/aggregation.


Loading on a gel
A freshly prepared reducing agent should be added (e.g. Dithiotreitol, final concentration 50 mM) to the volume prepared for loading. Heat at 70°C for 5 min, briefly spin down and load on a gel. Protein loads of 0.5-5 µg/lane should be sufficient for most Western Blot applications.


||| For other applications or usage on species other than stated above Agrisera offers a payment-after-testing option. To learn more about this or for any questions please use the LiveChat option or contact us at support@agrisera.com