What part of the plant can P-PER be used for?
Any part: stems, flowers, leaves, seeds, nuts (crack the outer shell and use the nut meat), and tree trunk (will need to grind before P-PER addition).
What is the ratio of P-PER Reagents to plant tissue?
For fresh/frozen samples – 1ml of aqueous (A+B) + 0.75mL organic (C) + 80mg fresh/frozen plant tissue
For dried samples/seeds – 1ml of aqueous (A+B) + 0.75mL organic (C) + 20mg dried plant tissue
Is mechanical abrasion necessary with P-PER?
Yes, you still need mechanical abrasion to disrupt the cell wall of plant cells. The mesh bag in the kit works well for soft plant tissues (e.g., leaves, flowers). You can use a pestle along with the mesh bag to disrupt harder tissues; place the tissue in the bag with the P-PER reagent, then tap the outside of the bag with the pestle.
Does the age and storage of the protein source affect protein yield?
Yes, as plants age their vacuoles (involved in cell maintenance) get larger. In older plants, 80% of the cell may be comprised of vacuoles. As vacuoles are a mostly liquid compartment, the amount of total protein is decreased as a plant ages. Protein can be extracted from fresh, frozen and dry plant tissue. If the sample has been powdered by freeze/grinding with liquid nitrogen, the extracted protein may no longer be active or useable in functional assays.
What protease inhibitor should I use?
You can use the same kind of protease inhibitor as is used for mammalian proteins. Although other suppliers sell plant protease inhibitors, there is nothing special about plant samples that require different inhibitors than for mammalian samples. Use EDTA-free Halt Protease Inhibitor (e.g., Product #78437) because EDTA soaks up the calcium. Calcium binds pectin, a sugar that binds proteins; hence, you cannot assay for these proteins. Thus the calcium is necessary to precipitate the pectin so that proteins are free for subsequent analysis. Note: Seeds have high amounts of proteases.
What phenolic inhibitor should I use?
Phenolics bind to proteins through hydrogen bonds immediately after plant cells are lysed. Thus it is important to use phenolic inhibitors; the two most common being PVPP and PVP. PVPP is the most widely used and is a generic phenolic inhibitor. It binds to phenolics through hydroxyl groups, but can also bind to hydroxyls on proteins resulting in protein loss. It is an insoluble material, like agarose, and takes hours (up to 24) to dissolve into solution. Recommended concentration is 1.5% w/v. PVP is a soluble substance and can be found in a variety of molecular weights. Smaller molecular weights can bind to proteins, so it is recommended to start with 2-4% 40KD PVP. Depending on the specific phenolics in the plant tissue, the molecular weight of PVP will need to be optimized. Different molecular weights bind certain phenolics better than others, so PVP does not bind all phenolics universally.
Can I prepare enough Working Solution to last for the week?
No. The P-PER Working Solution is prepared just before use. For best results, do not store the Working Solution for more than one day.
What if no separation of the phases is observed after Step 6?
Centrifuge the sample for an additional 5 minutes at 2,000 to 5,000 x g. If the tube is dropped after the phase separation, it will not harm the proteins to centrifuge again.
How can I determine the amount of protein extracted?
The P-PER Kit protein extracts can be quantified using the BCA Protein Assay Kit – Reducing Agent Compatible (Product # 23250).
Where are the nucleic acids after the procedure?
DNA is in the pellet after extraction. You can solubilize the pellet with sample buffer and run this sample on an agarose gel. Without shearing or a restriction digest, you will find genomic (large) DNA. You can use PEI (polyethyleneimine) to precipitate the DNA from any remaining proteins. One of our customers has used these nucleic acids for RT-PCR and was able to isolate RNA. It is recommended to wash the pellet twice with ice-cold Tris buffer and then dissolve in TE buffer.
What downstream applications is the aqueous buffer compatible with?
BCA Reducing Agent Compatible Protein Assay (Product #23250), 1-D gel electrophoresis, 2-D gel electrophoresis (dilute sample 1:10 in sample buffer), Western blot, immunoprecipitation, ion exchange chromatography, polyhistidine-tag protein purification, and activity assays.
How should I store my extracted proteins?
Once you have extracted the sample, do not keep the aqueous phase in contact with the organic phase for more than 30 minutes. If you do not plan to use the proteins immediately within 30 minutes, remove the aqueous phase and store at 4°C or -20°C. Specific applications may require protease inhibitors, phenolic inhibitors and reductants.
The Instructions recommend room temperature storage conditions for P-PER reagents. Would 4°C be better for long-term storage?
For P-PER Reagents A, B and C, room temperature storage is advised. Reagent C tends to become viscous at 4°C. Overall solubility of proteins and higher quality SDS-PAGE analysis are achieved with reagent C storage at room temperature. If the reagents need to be used at 4°C, place Reagents A,B,C at 0-4°C for 1-3 hours before use.