Proteins were extracted from frozen and homogenized seeds. For 4 mg seed material, 100 µL freshly prepared extraction buffer (4% (w/v) SDS, 2% (v/v) β-mercaptoethanol, 2 mM phenylmethane sulfonyl fluoride, 0.1 M Tris pH 8.5) was added. Samples were immediately, vigorously vortexed for at least 2 min. Afterwards, the samples were incubated at 80 °C for 3 min and centrifuged (10 min, 20,810 g, room temperature). The supernatant was transferred to a new tube and was mixed with 4 × Läemmli buffer. For SDS-PAGE and western blot analysis, 10 µL of with 4 × Läemmli buffer diluted protein extract was loaded on an SDS gel. For western blot analysis, proteins were detected using an anti-GFP antibody (diluted 1:5,000, BioLegend), monoclonal anti-c-MYC antibody (1:5000, Sigma) and monoclonal anti-FLAG M2 antibody (1:5,000, Merck) followed by the anti-Mouse IgG (whole molecule)-alkaline phosphatase (diluted 1:30,000, Merck). The SDS gel serving as loading control was stained with coomassie.
Anti mouse igg whole molecule alkaline phosphatase
Manufactured by Merck Group
Anti-Mouse IgG (whole molecule)-alkaline phosphatase is a laboratory reagent that consists of mouse immunoglobulin G (IgG) conjugated with the enzyme alkaline phosphatase. This conjugate can be used to detect the presence of mouse IgG in various immunoassay techniques.
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2 protocols using anti mouse igg whole molecule alkaline phosphatase
1
Protein Extraction and Analysis from Seeds
2
Protein Extraction and Western Blot Analysis
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Proteins were extracted from frozen and homogenized seeds. To 4 mg seed material, 100 µL freshly prepared extraction buffer (4 % (w/v) SDS, 2 % (v/v) β-mercaptoethanol, 2 mM phenylmethane sulfonyl uoride, 0.1 M Tris pH 8.5) were added. Samples were immediately vigorously vortexed for at least 2 min.
Afterwards, the samples were incubated at 80 °C for 3 min and centrifuged (10 min, 20810 g, room temperature). The supernatant was transferred to a new tube and was mixed with 4x Lämmli buffer. For SDS-PAGE and western blot analysis, 10 µL of with 4x Lämmli buffer diluted protein extract was loaded on a SDS gel. For western blot analysis, proteins were detected using an anti-GFP epitope tag antibody (diluted 1:5,000, BioLegend), monoclonal anti-c-MYC antibody (1:5,000, Sigma) and monoclonal anti-FLAG M2 antibody (1:5,000, Merck) followed by the anti-Mouse IgG (whole molecule)-Alkaline Phosphatase (diluted 1:30,000, Merck). The SDS gel serving as loading control was stained with coomassie.
Afterwards, the samples were incubated at 80 °C for 3 min and centrifuged (10 min, 20810 g, room temperature). The supernatant was transferred to a new tube and was mixed with 4x Lämmli buffer. For SDS-PAGE and western blot analysis, 10 µL of with 4x Lämmli buffer diluted protein extract was loaded on a SDS gel. For western blot analysis, proteins were detected using an anti-GFP epitope tag antibody (diluted 1:5,000, BioLegend), monoclonal anti-c-MYC antibody (1:5,000, Sigma) and monoclonal anti-FLAG M2 antibody (1:5,000, Merck) followed by the anti-Mouse IgG (whole molecule)-Alkaline Phosphatase (diluted 1:30,000, Merck). The SDS gel serving as loading control was stained with coomassie.
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