RbcL | Rubisco large subunit, form I (chicken)

AS01 017 | Clonality: Polyclonal | Host: Hen | Reactivity: [global antibody] for higher plants, algae, cyanobacteria | cellular [compartment marker] of plastid stroma

Product Information

Immunogen

KLH-conjugated synthetic peptide derived from all known plant,algal and cyanobacterial RbcL (Rubisco large subunit of Rubisco Form I) sequences, including Arabidopsis thaliana UniProt: O03042, TAIR: AtCg00490, Synechococcus sp. Q3ALL1

Host Chicken

Clonality Polyclonal

Purity Total IgY in PBS pH 8.0+ 0.02% sodium azide

Format Liquid

Quantity 50 µl

Storage Store at 4°C; make aliquots to avoid working with a stock. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from liquid material adhering to the cap or sides of the tubes.

Tested applications Immunolocalization (IL), ImmunoGold (IG), Western blot (WB)

Recommended dilution (IL) tested on a grass species, formaldehyde-fixed and paraffin-embedded tissue following the protocol from Gonzalez et al. (1998) Plant Physiol. V. 116. 1 : 10 000-1 : 20 000, 2 µg of total cellular protein, (WB)

Expected | apparent MW

52.7 kDa (Arabidopsis thaliana), 52.5 kDa (cyanobacteria), 52.3 kDa (Chlamydomonas reinhardtii)

Reactivity

Confirmed reactivity Arabidopsis thaliana, Fragilariopsis cylindrus, Lobaria pulmonaria, Medicago sativa, mixed phytoplankton, Pinus strobus, Pisum sativum, Solanum tuberosum, Spartina alterniflora, Spinacia oleracea, Synechococcus sp. PCC7842, Thiobacillus sp. Ulmus sp.

Predicted reactivity Algae, Dicots, Conifers, Liverworts, Mosses, Prochlorophytes, Welwitschia

Not reactive in No confirmed exceptions from predicted reactivity are currently known.

Application examples

Application examples Application example

western blot with chicken anti-RbcL antibodies

1 µg of total protein from samples such as Arabidopsis thaliana leaf (1) , Hordeum vulgare leaf (2), Zea mays leaf (3), Chlamydomonas reinhardtii total cell (4), were extracted with Protein Extraction Buffer PEB (AS08 300). 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 (GE RPN 2125; Healthcare) 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, 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 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.

Additional information

Peptide target used to elicit this antibody is not conserved in type II Rubisco found in dinoflagellates and some photosynthetic bacteria.

This antibody detects RbcL protein from 102.6 fmoles and has been used as a control to ensure adequate permeabilization and fixation of toxic cyanobacterial cells in immunolabeling experiments (method based on: Orellana & Perry (1995) J Phycol 31: 785-794).
Antibody has been used in immunolabelling of intact cyanobacterial cells fixed with ethanol using a secondary anti-IgY antibody conjugated with a fluorochrome.

For Rubisco quantification using quantitative western blot technique, anti-RbcL antibody, (AS03 037) combined with Rubisco ready to use standard (AS01 017) is recommended.

Related products

AS03 037 | Anti-RbcL | Rubisco large subunit, form I and form II (50 µl), rabbit antibodies
AS03 037A | Anti-RbcL | Rubisco large subunit, form I and form II (50 µg affinity purified), rabbit antibodies
AS03 037-HRP| Anti-RbcL | Rubisco large subunit, form I and form II (40 µg, HRP-conjugated), rabbit antibodies

AS15 2955 | Anti-RbcL II | Rubisco large subunit, form II (50 µl), rabbit antibodies
AS15 2955S | RbcL II | Rubisco form II positive control/quantitation standard

AS01 017S | Rubisco protein standard for quantitative western blot or positive control

AS03 037PRE | Rubisco large subunit, pre-immune serum
AS09 409 | Rubisco quantitation kit
AS15 2994 | Rubisco ELISA quantitation kit

AS07 218 | Anti-Rubisco | 557 kDa hexadecamer, rabbit antibody to a whole protein, rabbit antibodies

AS07 259 | Anti-RbcS | Rubisco small subunit (SSU), rabbit antibodies
AS07 222 | Anti-RbcS | Rubisco small subunit (SSU) from pea, rabbit antibodies

Recommended secondary antibodies:
Goat anti-chicken IgY (H&L), HRPconjugated or Goat anti-chicken IgY (H&L), ALP conjugated

Plant and algal protein extraction buffer

Background

Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the rate-limiting step of CO₂ fixation in photosynthesis. It is one of the most abundant proteins on Earth and its homology has been demonstrated from purple bacteria to flowering plants.