Protein phenol extraction
Protein purification by phenol extraction
Proteins (denatured) and lipids partition into phenol during aqueous phenol extraction. Contrary to popular molecular biology rumor, proteins are not in the interphase. Methanol (acetone) selectively precipitates proteins from phenol. A dense aqueous phase (added sucrose) puts the phenol layer on top and insolubles stabilize the interphase.
40% w/w sucrose
Liquid (88%) phenol, best quality by adding water to crystalline phenol
1% w/w Ficoll 400
10% w/v SDS
N-ethylmorpholine (NEM, liquid) or tris
All extractions at room temperature
Procedure - for minipurified samples
To 100ul of 40% sucrose in a 0.5ml conical plastic centrifuge tube add 150ul of phenol. Add minipurified virus (~20ul), 10%SDS (~10ul) and ME (~10ul). Mix thoroughly. Centrifuge 1 min (3K), draw off 100ul of upper phase into a 3ml Pyrex tube. Add 5ul of 1% Ficoll and 1ml of MeOH, mix, centrifuge 2min (5K). Discard supernatant, drain tube, wash ppt with MeOH (ppt sticks to tube) and dry. A hair drier speeds drying. Dissolve pellet in cracking buffer (~75ul).
Procedure - for plant tissue (3rd world friendly)
Grind 50mg of leaf in a mortar with 150ul of 40% sucrose, 500ul liquid phenol, 10ul 10%SDS, and 20ul ME (ME is important) (heating 3min at 70C did not improve extraction). Pour into a 0.5ml plastic centrifuge tube; centrifuge 1 min (3K), draw off 5ul (~1%) of upper phase into a 3ml Pyrex tube. Use a small volume here; large precipitates dissolve poorly. Add 5ul of 1% Ficoll (carrier) and 0.3ml of MeOH, mix, (incubation unnecessary) and centrifuge 3min (6K). Discard supernatant, drain tube, wash ppt with MeOH (ppt sticks to tube) and dry. A hair drier speeds drying. Dissolve pellet in cracking buffer (~75ul).
Maize leaves (with viruses) - Turnip leaves (with viruses)
Comparison minipurified virus vs. total leaf protein.
Simpler procedure - for plant tissue (3rd world friendly)
Cut leaf disk with #1 cork borer (smallest, dia ~4mm); grind in a mortar with 500ul of 40% sucrose, 10ul 10%SDS, 10ul ME (ME is important) and 50ul N-ethylmorpholine (equivalent to ~50mg of tris). Titrating phenol keeps DNA from the phenol phase. Pour into a 0.5ml plastic centrifuge tube, mix with 200ul liquid phenol (from crystalline); centrifuge 1 min (3K), draw off 30ul of upper phase into a 3ml Pyrex tube. Add 5ul of 1% Ficoll (carrier) and 0.3ml of MeOH, mix, (incubation unnecessary) and centrifuge 3min (6K). Precipitation is insensitive to MeOH volume. Discard supernatant, drain tube, wash ppt with MeOH (ppt sticks to tube) and dry. A hair drier speeds drying. Dissolve pellet in cracking buffer (~75ul), reduce and alkylate. Apply 2ul and 6ul samples to gels.
Tobacco samples - Aconitum samples - Brome mosaic - Hollyhock mosaic - Green tissue vs. white tissue
Phenol extraction of PBCV virion
Is a carrier necessary during MeOH precipitation?
Not if there's enough protein to see a precipitate
Scaling and other considerations - 0.1mm2 of leaf gives a reasonable protein pattern. One can visualize total protein from a single local lesion. With the cork borer protocol protein yield varies with species so adjust for leaf protein content by adjusting phenol volume. I typically mix aqueous reagents before extraction so a single pipeting suffices for each extraction. Chlorophyll is a good marker for the hydrophobic phase. I've used Ponceau S to mark the aqueous phase but found it unreliable. I extract in glass tubes to which protein precipitates adhere well. Avoid large amounts of protein because pellets are hard to dissolve. For small amounts of protein, acetone is a satisfactory (probably preferable to MeOH) precipitant.
Adding toluene to phenol phase decreases protein solubility - chlorophenol extracts proteins as well as (perhaps better than) phenol
Is it neutral pH or salt that's required for protein phenol extraction?
Acidic phenol extracts proteins efficiently if salt is present, however it also extracts acids which cause smearing on SDS gels.
Samples often have "junk" which interferes with SDS gel electrophoresis and protein detection. Mini-phenol extraction " clarifies" such samples. It selectively removes non-protein components in minipurified preps and clarifies samples from gum rich tissues. One can often detect virus inclusions among total leaf proteins. Total leaf protein analysis will miss many virus infections detectable by minipurification. However the technique is quicker and cheaper. It will likely detect some infections missed by other methods.
To ensure complete extraction heat a sample above 50C (where phases merge) to facilitate denaturation and mixing. Consider adding a standard protein as an internal control. If you see no proteins consider phase inversion (not enough sucrose to keep the phenol layer on top). Phases can be marked with lipid soluble or water soluble dyes.