Rubisco quantitation kit (Western blot)
This set enables quantification of Rubisco using Western blot in samples from a range of various species including dicots, monocots, algae, cyanobacteria.
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This set contains the following products:
AS03 037, RbcL | Rubisco large subunit, form I and form II (50 µl)
AS01 017S, Rubisco protein standard (100 µl)
AS08 300, Protein extraction buffer (2 x 2 ml)
AS09 602, Goat anti-rabbit IgG (H&L), HRP conjugated(2 x 10 µl)
AS16 ECL-N-10, AgriseraECL Bright (10 ml)
1 x 100 µl of AS01 017S, Rubisco protein standard (0.15 pmoles / µl, amount enough for generation of standard curve in 34 assays (standard curve: 0.0625 pmoles, 0.125 pmoles, 0.25 pmoles)
2 x 2 ml of AS08 300, Protein extraction buffer (amount enough for 48 isolations of plant material, using 500 µl 1x PEB for 100 mg fresh weight) or 120 isolations of algal material (using 200 µl 1x PEB for cell amounts corresponding to 4-10 µg total chlorophyll)
2 x 10 µl of AS09 602, Goat anti-rabbit IgG (H&L), HRP conjugated(amount enough for 50-100 Western blots)
1 X 10 ml of AS16 ECL-N-10, AgriseraECL Bright 10 ml (5 ml of component A + 5 ml of component B, trial pack)
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2 µg of total protein from various plant extracts (1-5) extracted with Agriera Protein Extraction Buffer PEB (AS08 300) separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Markers MagicMarks (Invitrogen) (M) and Rubisco protein standard (AS01 017S) at 0.0625 pmol, 0.125 pmol, 0.25 pmol.
Following standard western blot procedure this image has been obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad). The contour tool of the software is used to the area for quantitation and the values are background subtracted to give an adjusted volume in counts for each standard and sample.