For GUS staining, protein extracts were prepared from 10 mg of seedlings in 10 µl of SDS sample buffer. All of the eluate was loaded onto a 4–12% SDS-PAGE gel (Invitrogen) and the gel was run in a Bolt Mini Gel tank (Thermo Fisher Scientific). The resulting gel was subjected to immunoblot analysis using the iBlot2 dry blotting system (Thermo Fisher Scientific). Rabbit polyclonal anti-HSP22 (Eurofins Genomics) (1:1000 diluted), anti-HSP17.6 (ab80183; Abcam) (1:1000 diluted), and anti-HSP21 (ab80175; Abcam) (1:1000 diluted) and anti-rabbit IgG HRP conjugate (1:5000 diluted, Thermo Fisher Scientific) were used as primary and secondary antibodies, respectively. Signals were detected using chemiluminescence HRP substrates (Millipore) and an image analyser (LAS4000, GE healthcare). Protein size was determined by MagicMark XP (Thermo Fisher Scientific). Coomassie Brilliant Blue (CBB)-stained membranes were used as loading controls. The signal intensity of each band was quantified by ImageJ (NIH). Values in graphs are mean ± SEM. Three independent experiments were performed. Statistical significance was computed using a one-way ANOVA test followed by a post-hoc Tukey’s HSD test (https://astatsa.com/OneWay_Anova_with_TukeyHSD/ ).
Ab80175
Manufactured by Abcam
The Ab80175 is a polyclonal antibody that targets the Histone H3 protein. It is designed for use in various research applications, such as Western blotting, immunohistochemistry, and immunofluorescence. The antibody is produced in rabbits and purified using protein A affinity chromatography.
Automatically generated - may contain errors
Lab products found in correlation
Bolt mini gel tank, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg hrp conjugate, by Thermo Fisher Scientific (1 mentions)
Las4000, by GE Healthcare (1 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions)
Magicmark xp, by Thermo Fisher Scientific (1 mentions)
Sds page gel, by Thermo Fisher Scientific (1 mentions)
Chemiluminescence hrp substrate, by Merck Group (1 mentions)
Ponceau s, by Merck Group (1 mentions)
Protan nitrocellulose membrane, by Merck Group (1 mentions)
As06146, by Agrisera (1 mentions)
Pagerule plus pre stained protein ladder, by Thermo Fisher Scientific (1 mentions)
A11122, by Thermo Fisher Scientific (1 mentions)
Anti gfp antibody, by Roche (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
2 protocols using ab80175
1
Immunoblot Analysis of Heat Shock Proteins
2
Protein Extraction and Immunoblotting in Arabidopsis
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Total protein or soluble protein was extracted from Arabidopsis seedlings as described (Sedaghatmehr et al., 2016) . The protein concentration was assessed using the BCA Protein Assay kit (Thermo Fisher Scientific) or Bradford protein assay. Proteins were resolved by SDS-PAGE. The PageRule Plus Prestained Protein Ladder (10-250 kDa; Thermo Scientific) was used as the protein molecular weight marker.
For immunoblot analysis, proteins were blotted onto a Protan nitrocellulose membrane (Sigma-Aldrich, 10401396). Rabbit anti-HSP21 polyclonal antibody (Abcam, ab80175; 1:1000), rabbit anti-PsbD polyclonal antibody (Agrisera, AS06146; 1:5000), rabbit anti-HSP101 polyclonal antibody (Abcam, ab80121; 1:3000), and rabbit anti-green fluorescent protein (GFP) antibody (Invitrogen, A11122; 1:1000) were used. For autophagic flux experiments, we employed anti-GFP antibody from Roche (cat. no. 11 814 460 001). IRDye 800CW-conjugated goat antirabbit or anti-mouse IgG (H+L) antibodies were used as secondary antibodies at 1:10 000 dilution (LI-COR Biosciences). Detection of bands was done using the Odyssey Infrared Imaging System (LI-COR Biosciences). Co-IP assay was carried out as described, with 2% (v/v) Triton X-100 in the extraction buffer (Sedaghatmehr et al., 2019) . To demonstrate equal protein abundance, nitrocellulose membranes were stained with Ponceau S (Sigma).
For immunoblot analysis, proteins were blotted onto a Protan nitrocellulose membrane (Sigma-Aldrich, 10401396). Rabbit anti-HSP21 polyclonal antibody (Abcam, ab80175; 1:1000), rabbit anti-PsbD polyclonal antibody (Agrisera, AS06146; 1:5000), rabbit anti-HSP101 polyclonal antibody (Abcam, ab80121; 1:3000), and rabbit anti-green fluorescent protein (GFP) antibody (Invitrogen, A11122; 1:1000) were used. For autophagic flux experiments, we employed anti-GFP antibody from Roche (cat. no. 11 814 460 001). IRDye 800CW-conjugated goat antirabbit or anti-mouse IgG (H+L) antibodies were used as secondary antibodies at 1:10 000 dilution (LI-COR Biosciences). Detection of bands was done using the Odyssey Infrared Imaging System (LI-COR Biosciences). Co-IP assay was carried out as described, with 2% (v/v) Triton X-100 in the extraction buffer (Sedaghatmehr et al., 2019) . To demonstrate equal protein abundance, nitrocellulose membranes were stained with Ponceau S (Sigma).
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