Optimized Immunoblotting Protocol for Nif Protein Analysis

Immunoblotting was performed according to the method described previously (19 (link)). Briefly, samples were loaded on 10% SDS-polyacrylamide gels (Thermo Fisher Scientific; NP0301, NP0302) with 5 μL of PageRuler Prestained Protein Ladder (Thermo Fisher Scientific; 26616) as a marker. Proteins on the gels were subsequently transferred to PVDF membranes (Thermo Fisher Scientific; IB24002) using an iBolt 2 (Thermo Fisher Scientific). The membranes were blocked with 5% skim milk (BD Difco 232100; BD Biosciences) in PBS buffer and then incubated with primary antibodies for 4 to 8 h according to the sensitivity of the antibody for each Nif protein and labeled-tag. The secondary antibody goat anti-rabbit IgG-HRP (ZSGB Biotech; ZB-2301) or goat anti-mouse IgG-HRP (ZSGB Biotech; ZB-2305) was used at 1:3,000 dilution and incubated for 2 h before development. The primary antibodies used for immunoblotting and quantification in this study were as described previously (21 (link)), with the exception of different antibodies against GFP (ZSGB Biotech, TA-06), HSP60 (Proteintech, 66041-1-Ig) and Actin (HUABIO, HA601082).

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Yang J., Xiang N., Liu Y., Guo C., Li C., Li H., Cai S., Dixon R, & Wang Y.P. (2023). Organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria. Proceedings of the National Academy of Sciences of the United States of America, 120(34), e2305142120.

Publication 2023

NP0301 PageRuler Prestained Protein Ladder IB24002 iBolt 2 BD Difco 232100

Actin Anti igg Antibodies Buffer Dilution Gels Goat Membranes Milk Mouse Polyacrylamide gels Protein Pvdf Rabbit Sensitivity

Corresponding Organization :

Other organizations : Peking University, John Innes Centre

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Variable analysis

independent variables

Amount of sample loaded on the SDS-polyacrylamide gels
Duration of primary antibody incubation (4 to 8 hours)
Concentration of secondary antibody (1:3,000 dilution)

dependent variables

Protein levels of Nif proteins and labeled-tags detected by immunoblotting

control variables

Percentage of SDS-polyacrylamide gels (10%)
Protein ladder used (PageRuler Prestained Protein Ladder)
Type of PVDF membranes used (Thermo Fisher Scientific; IB24002)
Blocking solution (5% skim milk in PBS buffer)
Secondary antibodies used (goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP)
Primary antibodies used (as described previously in reference 21)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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