RbcL II | Rubisco large subunit, form II

AS15 2955 | Clonality: Polyclonal | Host: Rabbit | Reactivity: photosynthetic bacteria, archea, dinoflagellates

Benefits of using this antibody

RbcL II | Rubisco large subunit, form II

Product Information

Immunogen

KLH-conjugated synthetic peptide conserved in known RbcL form II protein sequences

Host Rabbit

Clonality Polyclonal

Purity Serum

Format Lyophilized

Quantity 50 µl

Reconstitution For reconstitution add 50 µl of sterile water

Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.

Tested applications Immunofluorescence (IF), Western blot (WB)

Recommended dilution

1:10 000 (IF), (WB)

Expected | apparent MW 51,7 kDa

Reactivity

Confirmed reactivity Alexandrium catenella, Amphidinium carterae, Chaetoceros neogracilis, Rhodobacter capsulatus, Rhodospirillum rubrum

Predicted reactivity Acidithiobacillus ferrooxidans, Dechloromonas aromatica, Gonyaulax polyedra, Leptothrix cholodnii, Magnetovibrio blakemorei, Rhodobacter sphaeroides, Thiobacillus denitrificans, Rhodopseudomonas palustris

and with a signle mismatch in one amino acid:
Gallionella capsiferriformans, Mariprofundus ferrooxydans, Thioalkalicoccus limnaeus, Methanomethylovorans hollandica, Methanococcoides burtonii, Methanosaeta concili, Methanolobus tindarius, Methanohalophilus mahii, and the dinoflagellate Symbiodinium sp. (ex Stylophora pistillata)
Species of your interest not listed? Contact us

Not reactive in Burkholderi, Cyanobium sp.

Application examples

Application example

western blot using anti-RbcL form II antibodies

1.5 µg of total protein extract from Rhodobacter capsulatus (1); extracted with Agrisera Protein Extraction Buffer PEB (AS08 300); 0.5 pmol of recombinant RbcL I (2), 0.5 pmol of recombinant RbcL II (3) Samples were diluted with 1X sample buffer (NuPAGE LDS sample buffer (Invitrogen) supplemented with 50 mM DTT and heat at 70°C for 5 min and keept on ice before loading. Protein samples were separated on 4-12% Bolt Plus gels, LDS-PAGE and blotted for 70 minutes to PVDF using tank transfer. Blots were blocked immediately following transfer in 2% blocking reagent or 5% non-fat milk dissolved in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 10 000 (in blocking reagent) for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, and then washed 1x15 min and 3x5 min with TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS10 1489, Agrisera) diluted to 1:25 000 in blocking reagent for 1h at room temperature with agitation. The blots were washed as above. The blot was developed for 5 min with chemiluminescent detection reagent of extreme femtogram sensitivity, according the manufacturers instructions. Images of the blots were obtained using a CCD imager (VersaDoc MP 4000) and Quantity One software (Bio-Rad). Exposure time was 30 seconds.

Western blot using RbcL II antibody on dinoflagelates

Western blot using RbcL II antibody on dinoflagelates

Quantitative Immunoblot of Form II RbcL from Amphidinium carterae ( Dinoflagellata ). Algae were extracted from PC filters in Agrisera Protein Extraction Buffer (AS08 300) with 3 breakage cycles (60 seconds, 6.5 m/s) in a FastPrep-24 with D-matrix ceramic beads (MP Biomedicals). Total algal protein extract (2 μg, lanes 1-8) and a range of loads of recombinant form II RbcL standard (AS15 2955S, lanes 9-13, 37.5, 75, 150, 300, 450 fmol) was separated on a Bolt polyacrylamide gel (ThermoFisher) and bloSed onto PVDF. Following antibody incubations (primary antibody AS15 2955, 1:20000; secondary antibody AS09 602, 1:20 000), protein signal was developed using chemiluminescence detection reagent and
visualised using a VersaDoc Imager (BioRad).

Courtesy of Environmental Proteomics N.B, Canada

Multiplexing using anti-RbcLII antibodies in diatom

Multiplexing using anti-RbcLII antibodies in diatom

Multiplex immunofluorescence detection of SxtA1 and RbcL, form II by confocal laser scanning microscopy in diatom Alexandrium catenella (Group I) as described in Cho et al. (2021).
Anti-RbcL form II antibodies were used in diltution of 1: 10 000.

Additional information

Additional information The antibody does not detect Form I RbcL/cbbL

Protein extraction from diatoms protocol can be found here.

Background

Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the rate-limiting step of CO2 fixation in photosynthetic organisms.

Form II Rubisco is present in many photosynthetic bacteria and archaea and in some photosynthetic dinoflagellates.

The large subunit (LSU) of form I Rubisco is encoded by the RbcL/cbbL gene, while form II LSU is encoded by cbbM.

Product citations

Selected references Cho et al. (2021). SxtA localizes to chloroplasts and changes to its 3'UTR may reduce toxin biosynthesis in non-toxic Alexandrium catenella (Group I). Harmful Algae, 2021,101972,ISSN 1568-9883, https://doi.org/10.1016/j.hal.2020.101972. Immunolocalization
Bausch et al. (2019). Combined effects of simulated acidification and hypoxia on the harmful dinoflagellate Amphidinium carterae. Marine Biology, June 2019, 166:80.
Long et al. (2018). Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts. Nat Commun. 2018 Sep 3;9(1):3570. doi: 10.1038/s41467-018-06044-0.

calculated \| apparent molecular mass [kDa]:	51,7 kDa
Clonality:	Polyclonal
Format:	Lyophilized
Host:	Rabbit
immunogen:	KLH-conjugated synthetic peptide conserved in known RbcL form II protein sequences
Purity:	Serum
Quantity:	50 µl
recommended dilution:	1:10 000 (IF), (WB)
Reconstitution:	For reconstitution add 50 µl of sterile water
storage:	Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
tested applications:	Immunofluorescence (IF), Western blot (WB)
additional information:	The antibody does not detect Form I RbcL/cbbL
additional information (application):	Protein extraction from diatoms protocol can be found here.
background:	Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the rate-limiting step of CO2 fixation in photosynthetic organisms. Form II Rubisco is present in many photosynthetic bacteria and archaea and in some photosynthetic dinoflagellates. The large subunit (LSU) of form I Rubisco is encoded by the RbcL/cbbL gene, while form II LSU is encoded by cbbM.
Picture (footer):	Application example 1.5 µg of total protein extract from Rhodobacter capsulatus (1); extracted with Agrisera Protein Extraction Buffer PEB (AS08 300); 0.5 pmol of recombinant RbcL I (2), 0.5 pmol of recombinant RbcL II (3) Samples were diluted with 1X sample buffer (NuPAGE LDS sample buffer (Invitrogen) supplemented with 50 mM DTT and heat at 70°C for 5 min and keept on ice before loading. Protein samples were separated on 4-12% Bolt Plus gels, LDS-PAGE and blotted for 70 minutes to PVDF using tank transfer. Blots were blocked immediately following transfer in 2% blocking reagent or 5% non-fat milk dissolved in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 10 000 (in blocking reagent) for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, and then washed 1x15 min and 3x5 min with TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS10 1489, Agrisera) diluted to 1:25 000 in blocking reagent for 1h at room temperature with agitation. The blots were washed as above. The blot was developed for 5 min with chemiluminescent detection reagent of extreme femtogram sensitivity, according the manufacturers instructions. Images of the blots were obtained using a CCD imager (VersaDoc MP 4000) and Quantity One software (Bio-Rad). Exposure time was 30 seconds. Quantitative Immunoblot of Form II RbcL from Amphidinium carterae ( Dinoflagellata ). Algae were extracted from PC filters in Agrisera Protein Extraction Buffer (AS08 300) with 3 breakage cycles (60 seconds, 6.5 m/s) in a FastPrep-24 with D-matrix ceramic beads (MP Biomedicals). Total algal protein extract (2 μg, lanes 1-8) and a range of loads of recombinant form II RbcL standard (AS15 2955S, lanes 9-13, 37.5, 75, 150, 300, 450 fmol) was separated on a Bolt polyacrylamide gel (ThermoFisher) and bloSed onto PVDF. Following antibody incubations (primary antibody AS15 2955, 1:20000; secondary antibody AS09 602, 1:20 000), protein signal was developed using chemiluminescence detection reagent and visualised using a VersaDoc Imager (BioRad). Courtesy of Environmental Proteomics N.B, Canada Multiplex immunofluorescence detection of SxtA1 and RbcL, form II by confocal laser scanning microscopy in diatom Alexandrium catenella (Group I) as described in Cho et al. (2021). Anti-RbcL form II antibodies were used in diltution of 1: 10 000.
All references:	Cho et al. (2021). SxtA localizes to chloroplasts and changes to its 3'UTR may reduce toxin biosynthesis in non-toxic Alexandrium catenella (Group I). Harmful Algae, 2021,101972,ISSN 1568-9883, https://doi.org/10.1016/j.hal.2020.101972. Immunolocalization Bausch et al. (2019). Combined effects of simulated acidification and hypoxia on the harmful dinoflagellate Amphidinium carterae. Marine Biology, June 2019, 166:80. Long et al. (2018). Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts. Nat Commun. 2018 Sep 3;9(1):3570. doi: 10.1038/s41467-018-06044-0.
Confirmed reactivity:	Alexandrium catenella, Amphidinium carterae, Chaetoceros neogracilis, Rhodobacter capsulatus, Rhodospirillum rubrum
predicted reactivity:	Acidithiobacillus ferrooxidans, Dechloromonas aromatica, Gonyaulax polyedra, Leptothrix cholodnii, Magnetovibrio blakemorei, Rhodobacter sphaeroides, Thiobacillus denitrificans, Rhodopseudomonas palustris and with a signle mismatch in one amino acid: Gallionella capsiferriformans, Mariprofundus ferrooxydans, Thioalkalicoccus limnaeus, Methanomethylovorans hollandica, Methanococcoides burtonii, Methanosaeta concili, Methanolobus tindarius, Methanohalophilus mahii, and the dinoflagellate Symbiodinium sp. (ex Stylophora pistillata) Species of your interest not listed? Contact us
not reactive in:	Burkholderi, Cyanobium sp.