Fluorescent protein-conjugated CRWNs were transiently expressed in Nicotiana benthamiana leaves by Agrobacterium infiltration. The leaves were sampled at 4 days after inoculation. Immunoprecipitation was performed with a µMACS GFP Isolation Kit (Miltenyi Biotec, Auburn, CA, USA). The leaves (1.0–2.0 g) were homogenized in µMACS lysis buffer (2.0–4.0 ml) containing Protease Inhibitor Cocktail Complete (Roche) and then the extracts were centrifuged at 10,000 × g for 10 min to obtain the soluble lysate. Anti-GFP antibody-conjugated magnetic beads were added to the lysate and the mixture was incubated at 4 °C for 30 min with gentle shaking. The GFP-conjugated proteins were isolated using a magnetic column, in accordance with the manufacturer’s instructions. The purified proteins were analyzed by western blotting using an anti-GFP antibody at 1:2000 (ab290; Abcam), an anti-tagRFP antibody at 1:500 (R10367; Thermo Fisher Scientific), and HRP conjugated anti-rabbit antibody at 1:10000 (458; MBL, Aichi, Japan)
R10367
Manufactured by Thermo Fisher Scientific
Sourced in Japan
The R10367 is a laboratory equipment designed for general laboratory use. It provides a stable and controlled environment for various laboratory applications. The core function of this product is to maintain temperature, humidity, and other environmental conditions as required for specific research or testing procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ab290, by Abcam (2 mentions)
A6455, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Macs gfp isolation kit, by Miltenyi Biotec (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (1 mentions)
Cm1900, by Leica (1 mentions)
Ab13970, by Abcam (1 mentions)
Fv1000, by Olympus (1 mentions)
D3571, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti chicken igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Amersham ecl prime detection reagent, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by Merck Group (1 mentions)
Amersham ecl prime, by Cytiva (1 mentions)
6 protocols using r10367
1
Affinity Purification of Fluorescent Proteins
2
Western Blot Analysis of Protein Samples
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Protein extracts were mixed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and denatured at 95 °C for 5 min. After brief centrifugation, denatured protein samples were subjected to 8 or 12% SDS-PAGE and blotted onto PVDF membranes. Primary and secondary antibodies were used at the following dilutions: 1:10,000 for GFP (ab290; Abcam); 1:10,000 for β-tubulin (MAB3408; Sigma-Aldrich); 1:5000 for RNAPII Ser2P (MABI0602; MAB Institute); 1:10,000 for RFP (R10367; Thermo Fisher Scientific); 1:10,000 for anti-IgG (rabbit) pAb-HRP conjugate (458; MBL); and 1:10,000 for anti-IgG (mouse) HRP conjugate (W402B; Promega). Immunoreactive bands were visualized by ImmunoStar® LD (Wako). The molecular weights were estimated with the ExPASy: SIB bioinformatics resource portal37 (link).
3
Immunostaining of Drosophila Larval Tissues
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Fat bodies or eye disks from wandering 3rd instar larvae were dissected in 1× PBS and fixed in 4% PFA, 1× PBS for 20 min. Tissues were then washed 1x with 1× PBS and permeabilized in 0.2% Triton-X 100, 1× PBS (PBST) for 20 min. The tissues were stained in PBST with primary antibodies at the indicated dilution and Alexafluor-conjugated secondary antibodies at 1:500 for 1.5 h, washed 3× with PBST and mounted with DAPI. All incubations were carried out at room temperature with mild agitation. Primary antibodies: Rabbit anti-GFP (1:500, Life Technologies A6455), Rabbit anti-RFP (1:250, Thermo Fisher R10367), Guinea pig anti-ATF4 (1:1054 (link)), Mouse anti-Rh1 (1:500, DSHB 4C5), Rabbit anti-PeIF2α (1:500, Cell Signaling 9721S).
4
Immunohistochemical Analysis of Spinal Cord
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After cardiac perfusion with 4% paraformaldehyde, the spinal cord was dissected and post-fixed, followed by cryoprotection in a graded series of sucrose solutions. Longitudinal sections (20 µm-thick) of the spinal cord were cut using a cryostat (CM 1900, Leica) and thaw-mounted onto Super Frost Plus slides (Thermo Fisher Scientific). For immunohistochemistry, sections were incubated overnight at 4℃ with the following primary antibodies: anti-RFP (1:500, mouse polyclonal; #R10367, Invitrogen), anti-adenomatous polyposis coli (APC) clone CC1 (APC-CC1) (1:200, mouse monoclonal; #OP80, Calbiochem), anti-glial fibrillary acidic protein (GFAP) (1:500, rabbit polyclonal; #Z0334, DAKO) and anti-doublecortin (DCX) (1:400, rabbit polyclonal; #4604S, Cell Signaling Technology). After washing, slides were incubated with appropriate secondary antibodies conjugated to the Alexa Fluor fluorescent dyes. Slides were examined using a confocal laser scanning microscope (LSM 800, Carl Zeiss).
5
Immunohistochemical Analysis of Larval Tissues
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Larvae were fixed in 4% PFA +3% sucrose for 2 h at RT followed by 3X 5 min washes in PBS. After removing the skin, larvae were embedded in 4% low melting agarose and cut in 50 μm sagittal sections. Sections were mounted on a slide and blocked for 1 h in blocking solution (10% NGS, 1% DMSO, 0.5% Triton X100 in 0.1 M PBS). Slides were incubated with primary antibody over night at RT or for two days at 4 °C (1% NGS, 1% DMSO, 0.5% Triton X100 in 0.1 M PBS). After washing three times for 5 min in 0.1 M PBS with 0.5% TritonX100 (PBST), slides were incubated in the dark with the secondary antibody in PBST. After washing again three times for 5 min in PBST, mounting medium was added and slices were imaged on a confocal microscope using a 63X objective (FV-1000, Olympus). Antibodies used here were Anti-GFP (Abcam ab13970, dilution 1:500), anti-TagRFP (Life Technologies R10367, dilution 1:500), Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen A11011, dilution 1:1,000), Alexa Fluor 488 goat anti-chicken IgG (Invitrogen A11039, dilution 1:1,000); all immunohistochemical samples were counterstained with DAPI (Invitrogen D3571, dilution 1:2,000).
6
Quantifying Rice Leaf Proteins
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Rice total protein was extracted (Chye et al., 1999 (link); Liao et al., 2014b (link)) from 1-week-old fresh rice leaves and protein concentration was measured by the Bradford assay (Bradford, 1976 (link)). Proteins (20 μg) were resolved on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Pall). Western blot analysis was performed as described previously (Lung et al., 2017 (link); Liao et al., 2018 (link)). The blots were cross-reacted with rabbit polyclonal anti-GFP (1: 5000, A6455; Invitrogen) for OsACBP4promoter:OsACBP4:GFP transgenic lines or rabbit polyclonal anti-RFP (1:7000, R10367; Invitrogen) for OsACBP5promoter:OsACBP5:DsRED transgenic lines at 4°C overnight, followed by incubation with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (1:50,000; Sigma-Aldrich) at room temperature for 1 h. Cross-reacting bands were detected by the Amersham ECL Prime Detection Reagent (GE Healthcare).
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