Whole cell lysates of each of the enriched Mod variants were prepared from mid-log (OD600 ∼ 0.4-0.7) cultures grown in BHI-NAD broth. Samples were normalized to OD600 of 2.0 and prepared for SDS-PAGE using Novex Bolt™ LDS sample buffer and β-mercaptoethanol (final concentration 10% [v/v]). Samples were boiled for 20 min at 95°C and centrifuged at 14 000 × g for 20 min. Samples were run at 150 V for 1 hr on Novex Bolt™ Bis–Tris Plus precast Gels (4–12%) with 1× MOPS buffer (Life Technologies Australia Pty Ltd) and transferred to a nitrocellulose membrane (Bio-Rad, California, United States) at 15 V for 90 min. The membrane was then blocked overnight with 10% (w/v) skim milk in 1× Tris-buffered saline with Tween 20 (0.05%) (TBST) with shaking. The membrane was washed with 1× TBST 3–5 times for a total of 20 min, then incubated with primary mouse antibody raised against TRD-less ModP and ModQ proteins at 1:1000 dilution for 1 h at room temperature. The membrane was washed 3–5 times with 1× TBST for a total of 20 min and then incubated for an hour with 1:3000 dilution of secondary antibody (goat anti-mouse alkaline phosphatase conjugate, Sigma). The membrane was washed again with 1× TBST 3–5 times for a total of 20 min and developed using 5-bromo-4-chloro-3-indolylphosphate (BCIP)/nitro-blue tetrazolium (NBT) (Roche) according to the manufacturer's instructions.
Goat anti mouse alkaline phosphatase conjugate
Manufactured by Merck Group
Goat anti-mouse alkaline phosphatase conjugate is a laboratory reagent used to detect and quantify the presence of mouse-derived proteins or antigens in samples. It consists of an alkaline phosphatase enzyme coupled to a goat-derived antibody that specifically binds to mouse immunoglobulins. This conjugate can be used in various immunoassay techniques, such as enzyme-linked immunosorbent assay (ELISA), to enable the visualization and measurement of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Mops buffer, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Goat anti rabbit alkaline phosphatase conjugate, by Merck Group (1 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (1 mentions)
Genetools software, by Syngene (1 mentions)
Bolt mops, by Thermo Fisher Scientific (1 mentions)
Sigmafast bcip nbt, by Merck Group (1 mentions)
Anti c myc antibody, by Merck Group (1 mentions)
Nbt and bcip, by Promega (1 mentions)
4 protocols using goat anti mouse alkaline phosphatase conjugate
1
Western Blot for Mod Variant Proteins
2
Western Blot Analysis of E7 and Zera® Proteins
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Samples were incubated at 85°C for 5 min in loading buffer, separated on 15% SDS-PAGE, then either stained with Coomassie blue or transferred onto a nitrocellulose membrane using the Trans-Blot® SD Semi-Dry Transfer Cell (Bio-rad) for western blot analysis. E7 proteins were detected with anti-E7 sera (1:4000) followed by goat anti-mouse-alkaline-phosphatase conjugate (1:10000; Sigma). Zera®-containing proteins were detected with polyclonal anti-Zera® sera (1:5000; ERA biotech) followed by goat anti-rabbit-alkaline-phosphatase conjugate (1:5000; Sigma). NBT/NCIP tablets (Roche) were used for final detection.
Proteins were quantified by measuring the density of the band on a western blot or Coomassie stained bands in comparison to a known protein concentration standard, using GeneTools software (SYNGENE) on scanned images. TSP was determined using the BioRAD assay according to the manufacturer's instructions.
Proteins were quantified by measuring the density of the band on a western blot or Coomassie stained bands in comparison to a known protein concentration standard, using GeneTools software (SYNGENE) on scanned images. TSP was determined using the BioRAD assay according to the manufacturer's instructions.
3
Western Blot Analysis of Lav-Bind Protein in NTHi
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Protein lysates of whole NTHi cells were prepared by heating whole-cell suspensions at 99°C for 40 min. These were electrophoresed on 4 to 12% Bis-Tris polyacrylamide gels (Invitrogen) at 150 V for 45 min in Bolt MOPS (morpholinepropanesulfonic acid) running buffer (Invitrogen). Samples were transferred to nitrocellulose membrane at 15 V for 1h. Membranes were blocked with 5% (wt/vol) skim milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) by shaking overnight at 4°C. Primary mouse antibodies against the Lav-bind protein (anti-Lav antisera) were raised in BALB/c mice at the Institute for Glycomics Animal Facility. Fifty micrograms of purified Lav-bind protein in alum was used per mouse. Primary antibody was used at a 1:1,000 dilution in 5% (wt/vol) skim milk in TBS-T for 1h with shaking at room temperature. Membranes were washed multiple times in TBS-T for 1 h before addition of secondary antibody (goat anti-mouse alkaline phosphatase conjugate; Sigma), as described above, at a 1:2,500 dilution. Membranes were washed for 1 h in TBS-T, before development at room temperature with SigmaFAST BCIP/NBT (5-bromo-4-chloro-3-indolylphosphate–nitroblue tetrazolium) prepared according to the manufacturer’s instructions (Sigma).
4
Quantifying Salinity-Induced TIR1 Protein Levels
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Seven dpg tir1-1 35S:TIR1-Myc plants were transferred to liquid ATS medium supplemented with 200 mM NaCl for different times. After treatments, plants were homogenized in ice-cold buffer [50 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS) pH 7.5, 200 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) Tween-20], containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and complete protease inhibitor cocktail (Roche) and centrifuged twice at 10,000 g at 4°C for 15 min. Equal amounts of protein were loaded onto SDS-PAGE and blotted onto the nitrocellulose membrane. Membranes were incubated with anti-c Myc antibody (Sigma) and goat anti-mouse alkaline phosphatase conjugate (Sigma) was used as secondary antibody. Then, Myc detection was visualized with NBT and BCIP (Promega). Densitometry analysis of immunoblots was performed using Matrox Inspector 2.2 software (Matrox Electronics Systems, Ltd).
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