| Additional information | | Suggested experimental treatments for student labs:
- Low light vs. High light
Shift from low or moderate light (50-100 umol photons m-2 s-1) to high light (500-2000 umol photons m-2 s-1, or nearly full sunlight).
Note: Depending on the species and the developmental stage this will lead to large changes in protein profiles, usually within a few hours and is reflected in detectable differences in LHC & RbcL content, and also differences in the PsbA/PsaC ratio (PSII:PSI).
-PsbA breakdown products will become more prominent, and the pool of mature PsbA usually declines
-there may be declines in antenna proteins, and sometimes increases in RbcL, if the stress is not too harsh.
For higher crop plants, it is probably the most generally reliable, rapid way to provoke detectable changes in protein content, since the PsbA/D1 pool will usually decline.
- Changes in nutritional status
Shifting from nutrient replete to low-nutrient conditions will cause gradual declines in LHC & RbcL content.
Etiolation vs. greening
Characteristic change, usually PSI (PsaC) accumulates before PSII (PsbA) and LHCII can be observed.
Developmental stages
Newly emerged, young & old leaves will show different profiles, as will (of course) leaves & roots. Suggested material:
Plants (spinach, sunflower, pea), Algae or cyanobacteria.
Material preparation and extraction:
Tissue samples of the size required for later extraction can be frozen in foil sachets and stored at –80ºC.
Extraction buffer:
LiDS is recommended over SDS for extraction involving cold steps.
Protease inhibitor: PEFABLOC. For a teaching lab, this may not be strictly necessary, however
if extracts are to be refrozen and reused, degradation becomes an issue and addition of protease inhibitor is advised.
Recommended extraction volume: 150 mg of leaf tissue into 500 ul of extraction buffer (see appendix). After the extraction by grinding and/or sonication the mixture is centrifuged (full speed in microfuge for 2 min) to separate solubilized extract from bulk leaf material. The recovery of extract should be about 300 ul, depending on the type of leaves. This gives an extract at a concentration of roughly 100 ug chl/ml (0.1 ug chl/ul).
Load per well: ca. 0.5 ug chl (so ca. 5 ul of the extract).
So 150 mg of leaf tissue should give a recovered extract of 300 ul @ 0.1 ug chl/ul = 30 ug chl, sufficient for about 60 lane loads of 0.5 ug chl/lane for immunoblotting
The loads can be reduced easily down to 0.2 ug chl (for mini gels) or even lower (down to 0.01 ug chl in some cases using ultrasensitive detection methods). Lighter loads are recommended for some abundant proteins detected with strong antibodies (like anti-RbcL).
Alternatively, for ease of extraction it can be scaled up to 1 g of tissue in 4 ml of extraction buffer. Loads can be prepared based on: chlorophyll, protein or leaf area.
Chlorophyll is suitable and simple loading parameter for determination of the level of the photosynthetic apparatus, which accounts for most leaf protein. Therefore loading by equal chlorophyll can show both qualitative and quantitative changes in a protein profile. For leaves, loads by equal area are recommended, as a leaf is nearly 2-dimensional and photosynthetic capacity per leaf area is a useful ecophysiological parameter.
Spectrophotometric chl determination
Extract chl from ca. 100 ul of aqueous extract into 900 ul of 80% acetone saturated with MgCO3.
Measure at A663 and A750 (Blank=1 ml 80 % acetone).
[Chla](μg.ml-1) = 12.7 (A663-A750)x 1000/x Quantitation
It is recommended to run 3 lanes of quantitated standard to generate a standard curve. It can be helpful to perform trial runs to get the quantitation standards and samples in the same range. Immunoblottting with primary and secondary antibodies and ECL shows linearity of detection over about 1 order of magnitude. Bands are detected over a much wider range, but the pseudo-linearity load/response region is only about 10 fold.
Agrisera Western blot protocols and additional information about antibodies |
