The reagent was described by Bradford, M., Analytical Chemistry: 72: 248-254, 1976. Mix 0.3 grams Coomassie Blue G-250 (not R-250, which is used for staining gels) in 150 ml absolute ethanol. Add 300 ml 85% phosphoric acid and mix thoroughly. Store refrigerated in 500 ml reagent bottles (we usually scale this up 3 or 4-fold). To prepare working reagent mix 150 ml stock with deionized water to final volume of 1 liter, filter twice through Whatman #1 qualitative paper (scale up if necessary). When mixing, start the stock solution stirring and slowly add water to final volume. Adding stock to water over-dilutes the reagent, which reduces its sensitivity to protein. Gradual dilution of the stock solution appears to give the most consistent results and the most sensitive dye reagent.
To kill motile cells and preserve flagella intact, a fixative consisting of 9% formaldehyde, 3% glutaraldehyde, 0.1% NaCl has given satisfactory results.
Per 10 ml, combine 2.4 ml 37.6% formaldehyde, 1.2 ml 25% glutaraldehyde, 170 µl 1M NaCl (10 mg), balance dH2O. Prepare and use this solution in the fume hood, keep fixed samples in capped Eppendorf tubes to minimize exposure to vapors.
Remove 100 µl Chlamydomonas suspension to an Eppendorf tube. Add 100 µl fixative, wait 2 min. before examining directly in a wet mount or adding LugolŐs iodine to stain flagella.
Prepare 6% KI, 4% I in dH2O, stir overnight wrapped in foil. Just prior to use, dilute one part dye with three parts Chlamydomonas medium, centrifuge, filter through 0.45 µm syringe filter.
Use of this fixative/stain renders flagella very easy to see in phase contrast or dark field modes. However, the iodine appears to cause cells to shed flagella as they die. Prior fixation with Chlamydomonas fixative may stabilize flagella and prevent shedding. Wait a minimum of 2 minutes before mixing fixed cells 50:50 with stain.
Prepare 100 mM stock phosphate solution by making up a stock of 100 mM sodium or potassium phosphate monobasic and one of dibasic. For sodium salts 100 mM monobasic stock consists of 12 gm/L anhydrous NaH2PO4 or 13.8 gm/L monohydrate NaH2PO4 . For dibasic stock use 14.2 gm/L anhydrous Na2HPO4. For potassium salts prepare 13.6 gm/L anhydrous KH2PO4 for monobasic stock, and either 22.8 gm/L K2HPO4o3H2O or 17.4 gm/L anhydrous K2HPO4 for dibasic stock.
To make a stock solution of desired pH fill a beaker 1/2 way with the dibasic solution (pH > 7) then add monobasic solution (pH < 7) while monitoring pH until the desired pH is reached.
Acrylamide stock (30%T, 2.5%Cbis) consists of 29.2% acrylamide, 0.8% bis-acrylamide in distilled water. The powders should be carefully weighed and mixed in a fume hood, with thorough cleanup afterwards. Mix well and filter one time through Whatman #1. Research grade acrylamide (2x crystallized) keeps well and is quite suitable. Serva is a good source. Finer grades (4x crystallized) deteriorate quicker and are more expensive. Store the stock solutions refrigerated in brown bottles.
Acrylamide is a cumulative neurotoxin. The polymer is safe, except for residual unpolymerized monomer. Limit contact, wear gloves, dispose as hazardous waste or (preferred) polymerize all leftover stock solutions, rinse, and throw away in regular trash. Dry chemicals are dusty, and a serious breathing hazard.
Provide students only with working quantities of stock solutions, to minimize the hazard. Leftover stock will be good later, but of course it may be alduterated by students.
To 3 L deionized water add 12.1 gm Tris, 57.6 gm glycine, while stirring. Add 4 gm SDS, bring to 4L (do not adjust pH). Store at RT in plastic jugs. We need about 600 ml per gel apparatus. This material does not keep well - electrode buffer supports bacterial and/or fungal growth. When contaminated buffer is used proteins enter the gels from the buffer, so that gel backgrounds do not destain.
Standards can be purchased from SIGMA in kit form. The MW-SDS-200 kit covers the range of 30,000 to 200,000 daltons, while MW-SDS-70L covers from 14,000 to 66,000. These are referred to as high and low molecular weight standards (HMW, LMW), respectively. The MW-SDS-70 kit, also available, is not suitable for the Laemmli system, on which this experimental design is based. One vial in each kit contains a lyophilized mixture of all of the standards. Add 1x sample buffer to the vial, following recommendations in flyer, and run on a gel to check staining intensity. The recommended loading amount is always too concentrated. We dilute the HMW mix (starting volume 1.0 ml) to 8 ml final volume with 1x sample buffer to get good bands. The LMW mix (starting volume 1.5 ml) is diluted to 6 ml final volume.
Standards are also provided in individual vials. Mix each with 1 ml 1x sample buffer and run several dilutions of each standard on appropriate gels to determine staining intensity. Prepare mixtures by combining concentrated stocks and bringing to volume with 1x sample buffer. Note that they are very stingy with myosin heavy chain (0.5 mg), which requires the least amount of dilution.
Per 100 ml desired, mix:
25 ml 4 x concentrated SDS-PAGE stacking gel buffer
4.6 g Sodium dodecyl sulfate (SDS; lauryl sulfate)
20 ml Glycerol
1.23 g Dithiothreitol (DTT; Cleland's reagent) - NOTE - Cleland's deteriorates with aged, even when frozen. Buffer should be made fresh.
dH2O to 100 ml
Heat the mixture in the microwave to dissolve SDS, then add 1 "pinch" Bromophenol blue dye - about 5 mg. Store below 0ű in aliquots of 1 ml in Eppendorf tubes. Avoid repeated freezing and thawing and prolonged storage. Old buffer can be used, but DTT should be replenished (1 gm per 100 ml). Insufficient DTT (reducing agent) is evident by non-reproducible patterns of bands in well-characterized samples, especially membrane-associated samples.
Separating gel buffer, 4 x concentrated stock, consists of 1.5 M Tris-Cl, pH 8.8, 0.4% SDS (181.7 g Trizma base, 750 ml dH2O, 4 g SDS, pH to 8.8 with concentrated HCl, dH2O to 1000 ml). Use a calomel pH electrode suitable for Trizma solutions or use a specific pH standard, since many conventional electrodes misread the pH of Tris solutions. Also note that the pH of a Tris solution is concentration-dependent. If one "overshoots" while adjusting pH, it is not advisable to raise pH back up with base. Potassium salts, in particular, must be avoided, as potassium precipitates SDS. Store at RT.
Stacking gel buffer, 4 x concentrated stock, consists of 0.5 M Tris, pH 6.8, 0.4% SDS (60.6 gm Trizma base, 750 ml dH2O, 4 g SDS, pH to 6.8 with concentrated HCl, dH2O to 1000 ml). Refer to instructions for separating gel stock for precautions.
Prepare 0.1% Coomassie blue R-250 (not G-250) in methanol:acetic acid:deionized water; 5:1:4. Make a gallon (3.8 liters) at a time. The working solution should be filtered before use through Whatman #1, storage at RT in a flammable cabinet. The stain can be reused, but as it becomes contaminated with SDS the staining effectiveness is reduced. Life of the stain can be prolonged by rinsing gels before adding the staining solution.
Wright's stain can be purchased as
Wright's quick stain from suppliers
(preferable), or prepared from powder.
From powder, mix 0.3 gm powder with
3 ml glycerol in a mortar. gradually
add acetone-free methanol to final volume
100 ml. Mix well, store tightly stoppered.
Let stand with occasional shaking for
at least one week, and filter immediately
before use. Wright's stain buffer is
prepared as 6.63 g potassium phosphate
monobasic (anhydrous) and 2.56 g sodium
phosphate dibasic (anhydrous) per liter.