Protocols > Extraction of Leaf Proteins

Protocol from Dr. Alice Barkan Lab, University of Oregon, USA

• KEEP EVERYTHING COLD AT ALL TIMES
• DO NOT ALLOW LEAF SAMPLES TO COME CLOSE TO THAWING UNTIL GRINDING IN HOMOGENIZATION BUFFER.
• KEEP EXTRACTED SAMPLES OUT OF THE –80°C FREEZER FOR THE MINIMAL AMOUNT OF TIME TO AVOID PROTEIN DEGRADATION. Remove samples from freezer immediately prior to use (i.e. after tubes with SDS-sample buffer are labelled and prepared) and return immediately after pipetting out aliquots for analysis. If extracting many samples, extract in batches of six, spot onto Coomassie for quantification (see below) and place in -80°C freezer before extracting the next batch. When experienced, it should be possible to do 18 preps in 30 min. When starting out, plan to do 12 preps in 30 min, in two groups of six.
• Protein Quantification: If care is taken when harvesting tissue to always harvest the same amount (e.g. a 1 cm leaf tip of the 2nd leaf), there is no need to quantify protein by Coomassie spotting. Otherwise, use the simple quantification procedure outlined below.
• Extract protein from several wild-type seedlings that were grown in parallel with each batch of mutants. The same wild-type controls can be used for all plants that were planted and harvested at the same time and place.

Procedure

1. Make a list of samples that will be extracted

Label 1.4 ml Eppendorf tubes with fine-tipped black sharpie (top: seedling number and phenotype (e.g. F235-7-2 pg) , side: seedling number, date (month/year). It is important to number on top and side because numbers can rub off.

Gather materials: mortars and pestles (6 small ones), liquid nitrogen (if working with frozen samples), protein extraction buffer (see below), gray Eppendorf tube rack with ice, Whatman paper for spotting to quantify protein; label with pencil the positions where each sample will be spotted.

2. Prepare homogenization buffer

Add the labile additives described below (ß-ME, PMSF, leupeptin and pepstatin) to a 5 ml aliquot of the Homogenization Buffer Base Stock. This will be sufficient to prep ~ 20 samples. Store the complete buffer on ice and discard unused buffer after 30 min.

Protein Homogenization Buffer Base Stock (store at 4°):

Before use, aliquot sufficient buffer for 30 min of extractions (e.g. 5 ml) and add:

• PMSF to 2 mM (1/20th volume of a 40 mM stock in isopropanol stored at –20°C. The stock should be mixed and heated sufficiently to put any crystals into solution before pipetting. PMSF is toxic and inactivates within 30 min after adding to aqueous solution).
• ß-mercaptoethanol (ß-ME) to 40 mM. (27 µl of the pure liquid stock (stored in hood) per 10 ml)
• Pepstatin to 2 µg/ml. (1/500th volume of a 1 mg/ml stock. The stock is stored at –20°C.)
• Leupeptin to 2 µg/ml (1/5000th volume of a 10 mg/ml stock. The stock is stored at –20°C)

For 5 ml of complete buffer add: 13 µl B-ME, 10 µl 1 mg/ml pepstatin, 250 µl 40 mM PMSF and 1 µl 10 mg/ml leupeptin. Discard this buffer after 30 min.

3. Grind tissue

Grind in small mortar and pestle.

If using fresh tissue, it is simplest to grind the unfrozen tissue directly in ice-cold homogenization buffer (200 µl) in pre-chilled mortar and pestle.

If using frozen tissue, grind tissue to a fine powder in liquid nitrogen and then add 200 µl homogenization buffer to the frozen powder and continue grinding to a paste. It is essential to keep the tissue frozen until the buffer is added.

Must grind hard to break bundle sheath cells. Failure to do this will reduce the yield of Rubisco and other bundle sheath-specific proteins.

4. For Total Leaf Extract (the usual case for routine mutant screening), pipet the extract into the labelled Eppendorf tube, on ice

If quantifying by Coomassie spotting (i.e. if the amount of tissue harvested was not similar for all samples), spot 2 µl onto Whatman 3MM paper (label spot with pencil).

For analysis of separated membrane and soluble proteins: pellet the membranes by microfuging for 3 min at top speed in the microfuge in the cold; pipet off the sup avoiding the green pellet, and place in a new microfuge tube. This is the "soluble" fraction. Rinse the membranes to be sure they are free of soluble proteins: Add 0.2 ml homogenization buffer to each membrane sample, flick gently with your finger, and microfuge for 2 min. Discard the sup; resuspend the membrane pellets in 50-200 µl homogenization buffer, by pipetting up and down gently. For a series of samples that you want to compare on a gel, resuspend the smallest green pellet in 50 µl, and the remaining pellets in whatever volume it takes to give an equal density of greenness (to your eye).

5. Store the protein samples in the -80°C freezer

Repeated freeze-thaw cycles can lead to protein degradation. So for particularly precious samples, freeze them in small aliquots.

6. Quantify the protein

If samples contained uneven amounts of leaf tissue, the relative protein concentrations can be estimated by the following simple procedure.

Spot 2 µl of each sample onto Whatman 3MM (with samples labelled).

To aid in quantification, spot 1:2 dilutions of several samples that you expect are particularly concentrated (i.e. they are particularly green). Each spot should have the same volume- i.e. on piece of parafilm, mix 2 µl of sample to 2 µl water, and then spot 2 µl of this onto the Whatman paper. This will allow you to see what 1/2 as much protein looks like. For even better quantification, spot 1, 3 and 10 µg of BSA in a 2 µl volume.

Place the Whatman 3MM paper with the protein spots in a container that has previously been used for Coomassie staining (i.e. it is stained blue). Add the Coomassie staining solution (325 ml water, 125 ml isopropanol, 50 ml acetic acid, 250 mg Coomassie Blue R-250). Rock briefly until you can see the spots turn blue (10-20 seconds). Rinse with water until the background is fairly white. Compare signal intensities while paper is either fully wet or fully dry. If it is partially dry, results may be misleading.

Gels- flick tube to suspend stuff. Pipet out what you want to put in sample, heat to 70ish, 1 min spin.

Important notes:
Proteins should be extracted very promptly, in the cold and for some proteins dark conditions have to be applied (like PIF proteins). Bead beater will allow parallel extraction of multiple samples. Recommended conditions for leaf material are for example: 2 mL tubes with 1 or 2 metallic beads with 30/s frequency for 30 s.