RbcL | Rubisco large subunit, form I (affinity purified)

Rubisco quantitation in plant and algal samples using Agrisera anti-RbcL antibody and Rubisco protein standard

Methodology: Plant samples are generally ground with liquid nitrogen in a mortar and pestle. The resulting powder is transferred to a plastic tube. Algal samples can be either concentrated by centrifugation or, preferably, by filtration onto glass fiber filters. Solubilization is performed in Agrisera protein extraction buffer (PEB, AS08 300) containing 0.1mg/mL PefaBloc SC (AEBSF) protease inhibitor (Roche). Disruption is most optimally obtained through flash freezing of the sample in liquid nitrogen alternated with thawing by sonication with a microtip. This process can be repeated depending on the toughness of the sample. The sample is adjusted to 50 mM dithiothreitol and heated to 70°C for 5 minutes. Samples are cooled and centrifuged briefly prior to electrophoresis.

Optimal quantitation is achieved using moderate sample loads per gel lane, generally 0.5 to 2.5 ug total protein, depending on the abundance of the target protein.

Electrophoresis and Immunoblotting: Once solubilized, the proteins can be separated electrophoretically in a number of systems. We obtain optimal results with the Invitrogen NuPAGE gel system using Bis-Tris 4-12% gradient gels. Proteins are separated in MES SDS running buffer according to the manufacturer’s recommendations at 200 V for 35 minutes. The gels are transferred to PVDF in the same apparatus, the SureLock XCell blot module, for 60 minutes at 30 V for a single gel or 80 minutes for a pair. Following transfer the blots are blocked with non-fat dry milk up to 10 % in TBS-T, for 1 h/RT with gentle agitation. The blot is incubated with primary antibody, usually at 1:25 000 to 1:50 000, for 1 h/RT (if extreme femtogram detection reagents are used) or in lower primary antibody dilution for less sensitivie reagents (mid picogram and lower).

For quantitation a relatively high primary antibody: target protein ratio gives more reliable results than immunoblots at low ratios of primary antibody:target protein.

The blot is washed extensively in TBS-T (twice briefly, once for 15 minutes and three times for five minutes). The blot is incubated with secondary antibody, for example goat anti-rabbit IgG horse radish peroxidase conjugated, AS09 602 (Agrisera), for 1h/RT. The blot is washed as above and developed with ECL detection reagents.

Quantitation: When quantitated standards are included on the blot, the samples can be quantitated using the available software. Excellent quantitation can be obtained with images captured on the Bio-Rad Fluor-S-Max or equivalent instrument using Bio-Rad QuantityOne software. The contour tool is used to select the area for quantitation and the values are background subtracted to give an adjusted volume in counts for each standard and sample. Using above protocol linear standard curves are generated over 1-1.5 orders of magnitude range in target load. It is important to note that immunodetections usually show a strongly sigmoidal signal to load response curve, with a region of trace detection of low loads, a pseudolinear range and a region of saturated response with high loads. For immunoquantitation it is critical that the target proteins in the samples and the standard curve fall within the pseudolinear range. Our total detection range using this protocol spans over 2 orders of magnitude, but the quantifiable range is narrower.

References:

MacKenzie et al  (2005). Large reallocations of carbon, nitrogen and photosynthetic reductant among phycobilisomes, photosystems and Rubisco during light acclimation in Synechococcus elongatus are constrained in cells under low environmental inorganic carbon. Arch of Microbiol. 183: 190 - 202.
Bouchard et al. (2006) UVB effects on the photosystem II-D1 protein of phytoplankton and natural phytoplankton communities. Photochem and Photobiol 82: 936-951. 
Morash et al. (2007) Macromolecular dynamics of the photosynthetic system over a seasonal developmental progression in Spartina alterniflora. Can J. of Bot. 85: 476-483(8)

Application example

2 µg of total protein from (1-7) Prochlorococcus sp. extracted with Protein Extration Buffer, PEB (AS08 300), (8-11) Rubisco protein standard (AS01 017S) were separated on  4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer for 1h/RT with agitation. Blots were incubated in the primary antibody at a dilution of 1: 20 000 for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:50 000 for 1h/RT with agitation. The blots were washed as above and developed for 5 min with ECL detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad). There is a low level of a known RbcL cleavage product below the main band in som eof the Prochlorococcus samples.

Recommended secondary antibodies: goat anti-rabbit HRP conjugated, goat anti-rabbit ALP conjugated

Recommended chemiluminescent detection reagent: AgriseraECLBright

 Method for embedding in LR WHITE for immunolabelling using anti-RbcL antibody 

Prins et al. (2008). Cysteine proteinases regulate chloroplast protein content and composition in tobacco leaves:a model for dynamic interactions with ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) vesicular bodies. J.Ex.Bot.59:1935-1950. Method described below is a courtesy of Dr Aranda

Samples must be sectioned at 1 x 1 mm. 1)Fixation: Glutaraldehyde 0.5% + paraformaldehyde 2.5% in sodium phosphate buffer 0.1M pH= 7.2, 1 hour and 30 min. 2) 3 washes in sodium phosphate buffer 0.1M pH= 7.2 (15 min. each one).  3)Dehydratation and embedding:

-35% Ethanol……30 min.
-50% Ethanol……30 min.
-70% Ethanol……30 min.
-96% Ethanol……30 min.
-100% Ethanol……30 min.
-100% Ethanol……30 min.
-100% Ethanol……30 min.

-75% Ethanol + 25% LR White…… 1 hour
-50% Ethanol + 50% LR White…… 1 hour
-25% Ethanol + 75% LR White…… 1 hour
-100% LR White…………………... 1 hour
-100% LR White…………………... overnight.

4) Polymerization: Oven 12-18 hours at 50ºC. For flat molds in continous flux of N2

5) Block sectioning: Ultrathin sections (60-70nm) using a ultramicrotome. Ultrathin sections should be placed on formvar coated níkel grids.

 IMMUNOLABELLING

1)Blocking: Place grids with sections into 30-μL drop of blocking buffer during 30 minutes. (Blocking buffer: phosphate-buffered saline (PBS) + 5% bovine serum albumin (BSA).

2) Incubation: Incubate in drop of primary antibody, anti-RbcL (Rubisco form I and II, Agrisera) diluted 1:250 in blocking buffer. 3 hours at room temperature (could be extend to overninght).

3) Rinse: Pass the grids through a series of drops of PBS + 1%BSA. Two or three changes during 5 minutes each one.

4) Incubation: Incubate grids in 30-μL of secondary antibody gold-labelled (British BioCell International, 10nm), diluted 1:50 in blocking buffer during 1.5 hours at room temperature.

5) Rinse: Pass the grids through a series of drops of PBS + 1%BSA. Two changes during 5 minutes each one.

6) Rinse: Pass the grids through a series of drops of PBS. Two or three changes during 5 minutes each one.

7) Rinse: Pass the grids through a series of drops of water (ultra-pure water must filtered 0.2 μm) . Four to five changes during 5 minutes each one, allowed to dry for later observation.

8) Staining: The grids may be post-stained with uranyl acetate and lead citrate.

Pao et al. (2018). Lamelloplasts and minichloroplasts in Begoniaceae: iridescence and photosynthetic functioning. J Plant Res. 2018 Mar 2. doi: 10.1007/s10265-018-1020-2. (ImmunoGold)
Liang et al. (2014). Cyanophycin mediates the accumulation and storage of fixed carbon in non-heterocystous filamentous cyanobacteria from coniform mats. PLoS One. 2014 Feb 7;9(2):e88142. doi: 10.1371/journal.pone.0088142. eCollection 2014. (ImmunoGold)