The micro-sized cell fractions were separated by SDS-PAGE (12%), and immunoblots were conducted as described23 (link). Protein was quantified by using the bicinchoninic acid (BCA) assay90 (link). Antibodies against LHCSR3 (AS142766, Agrisera), LHCSR1 (AS142819, Agrisera), and Psab (AS10695, Agrisera) were used at 1:5000 dilution. The D1 antibody was a gift from Guangye Han (Institute of Botany, Chinese Academy of Sciences), and the Lhcb1 antibody from Zhenfeng Liu (Institute of Biophysics, Chinese Academy of Sciences). Both antibodies were used at 1:1000 dilution. The blots were imaged using a chemiluminescence imaging system (BLY-9650, BOLAIYAN).
As10695
Manufactured by Agrisera
AS10695 is a laboratory equipment product. It is designed to perform a specific core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
As142766, by Agrisera (1 mentions)
As142819, by Agrisera (1 mentions)
As05085, by Agrisera (1 mentions)
Immobilon pvdf membrane, by Merck Group (1 mentions)
As09 602, by Agrisera (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
As11 1787, by Agrisera (1 mentions)
As06146, by Agrisera (1 mentions)
4 protocols using as10695
1
Immunoblotting of Micro-Sized Cell Fractions
2
Protein Separation and Western Blot Analysis
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The samples for Western blot analysis were first subjected to SDS-PAGE using 12% acrylamide gel with 6M urea to separate proteins according to size. The samples were loaded based on the Bradford assay to 1–4μg protein per well (see each experiment for details), with 1:1 vol buffer containing 138mM Tris–HCl pH 6.8, 6M Urea, 22.2% glycerol, 4.3% SDS, 10% β-mercaptoethanol, and a small volume of Bromophenol blue. The separated proteins were electro-transferred from the acrylamide gel to Immobilon PVDF membrane (Millipore). The PVDF membrane was blocked with 5% milk (BIO-RAD blotting grade blocker) and incubated with first antibody and second antibody and finally with ECL™ Western blotting Detection Reagent (Amersham™ GE Healthcare) before imaging on film. The protein-specific antibodies used in the assay were α-ADO (custom made, see Generating ADO-specific antibody above) at 1:1000 dilution, α-PsaB (AS10 695, Agrisera) at 1:1000 dilution, and α-ATPase β (AS05 085, Agrisera) at 1:5000 dilution. The secondary antibody (AS09 602, HRP conjugated Goat anti-Rabbit IgG (H&L), Agrisera) was used at 1:10000 dilution.
3
Chlamydomonas Protein Extraction and Detection
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Cell breakage and isolation of total protein and protein content measurements were performed as described (Ossenbuhl et al., 2004 (link)). The Chlamydomonas antibodies against PsbD (1:10,000), PsbC (1:5,000), PsaB (1:5,000) were from Agrisera (AS06146, AS111787, and AS10695) and diluted according to the supplier’s instructions. The antibodies against RbcL (1:5,000), Orf1995 (1:1,000), LHCII (1:5,000), HSP70D (1:10,000), HSP70B (1:5,000), ATG3 (1:5,000), VIPP1 (1:5,000), and RPL37 (1:10,000) were used as previously reported (Ramundo et al., 2013 (link), 2020 (link)). Antibodies against CPN60α (1:5,000), CPN60β1 (1:5,000), CPN60β2 (1:5,000), CLPT (1:1,000), and CLPR6 (1:1,000) were from Professor Cuimin Liu. The antibody against ATG8 (1:1,000) was from Professor Faqiang Li. The antibody against TOR1 (1:5,000) was from Professor Wenfei Wang. The antibody against HSP22E/F (1:1,000) and VIPP2 (1:5,000) was from Silvia Ramundo. Protein separation by SDS–PAGE and immunoblot analysis were performed as described previously (Ramundo et al., 2013 (link)).
4
Protein Extraction, Electrophoresis, and Immunoblotting
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Total protein extraction, electrophoresis, and immunoblotting were performed as described previously (Huokko et al., 2019) . Antibodies raised against PsaB (AS10 695, Agrisera), D1 (AS11 1786, Agrisera), and Flv3 (Antiprot) were used in this study.
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