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).
Np0301
Manufactured by Thermo Fisher Scientific
The NP0301 is a lab equipment product from Thermo Fisher Scientific. It is a device used for general laboratory applications. The core function of the NP0301 is to provide a controlled environment for various experiments and analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Pageruler prestained protein ladder, by Thermo Fisher Scientific (2 mentions)
Ibolt 2, by Thermo Fisher Scientific (2 mentions)
Difco 232100, by BD (2 mentions)
Ib24002, by Thermo Fisher Scientific (1 mentions)
Nupage gel system, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
3 protocols using np0301
1
Optimized Immunoblotting Protocol for Nif Protein Analysis
2
Protein Immunoprecipitation and Mass Spectrometry
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Cells were washed 2x in ice cold PBS, then lysed in lysis buffer (10% Glycerol, 50 mM HEPES-KOH pH 8.0, 100 mM KCl, 2 mM EDTA, 0.1% NP-40, 1x Protease inhibitor (Sigma #), 10 mM NaF, 0.25 mM NaOVO3, 5 nM Okadaic acid, 5 nM Calyculin A, 50 mM β−glycerolphosphate) for 60 minutes with intermittent vortexing. Lysates were freeze-thawed at −80C, thawed on ice, then centrifuged at >15,000 RPM for 60 minutes. Each supernatant was transferred to a new tube, and spun >15,000 RPM for additional 30 minutes. Protein supernatants were incubated with 10μg of antibody overnight at 4 C on a nutator. Immunocomplexes were precipitated with Protein G (for IgGs), FLAG-M2 (FLAG-tagged Fabs) or streptavidin beads (Avi-tagged Fabs), as indicated (beads purchased from Pierce, Cat# 53125, Sigma, Cat# A2220 and Pierce, Cat# 20347, respectively). Beads were washed well with lysis buffer, followed by H2O. Proteins were eluted in SDS-PAGE sample buffer (NuPage gel system, ThermoFisher, Cat# NP0301) for western blot, or processed as below in 2.4.8 for mass spectrometry (MS).
3
Immunoblotting Analysis of Nif Proteins
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Samples were loaded on 10% SDS-polyacrylamide gels (Thermo Fisher Scientific; NP0301, NP0302) with 6 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 the iBolt 2 (Thermo Fisher Scientific). The membranes were blocked with 5% skim milk (BD Difco 232100; BD Biosciences) in PBS buffer. The antibodies for each Nif protein were used at a dilution of 1:500 to 1:3,000 (according to the sensitivity of the antibody for each Nif protein). 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. Development was done by using two different methods: using an enhanced chemiluminescent substrate for HRP (Thermo Fisher Scientific; 34077) and captured by an automatic chemiluminescence imaging analysis system (Tanon-4200) or directly developed on the PVDF membranes using the DAB Chromogen/HRP Substrate Kit (ZSGB Biotech; ZLI-9018). The antibodies used for immunoblotting and quantification in this study were as described previously (36 (link)).
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