Fluorescent proteins were detected from whole cell lysates. S. aureus was cultured overnight as described in the growth conditions. The pre-cultured cells were adjusted to an optical density at 660 nm of 0.02 with TSB with chloramphenicol, and 3 ml of the culture were transferred into test tube (25 mm × 150 mm) and then incubated at 37 °C with shaking at 140 rpm for 16 h. The bacterial cells from 1 ml cultures were harvested by centrifugation, and then the whole cell lysates were prepared as follows: cells were re-suspended in 200 μl CS buffer (100 mM Tris-HCI, 150 mM NaCl, 100 mM EDTA, pH7.5) containing 1 μg of lysostaphin (Wako Pure Chemical Industries, Co., Ltd, Japan) and incubated at 37 °C for 30 min. Ten microliters of cell lysates were separated on SDS–PAGE and stained with Coomassie Brilliant Blue (CBB), and the fluorescent proteins were detected using Western blot with the following antibodies: Anti-GFP-HRP-Direct T (MBL Co., Ltd.), Living colors® Anti-RCFP Polyclonal Pan Antibody (Clontech Laboratories) and DsRed Polyclonal Antibody (Clontech Laboratories) as the primary antibody, and HRP-conjugated goat antibodies against rabbit IgG (MP Biomedicals, LLC-Cappel Products) as the secondary antibody. Immuno-detection of protein was performed on Pierce® Western Blot Substrate (Thermo Fisher Scientific Inc.) with X-ray film.
Dsred polyclonal antibody
Manufactured by Takara Bio
The DsRed polyclonal antibody is a research tool used to detect and localize the DsRed protein in various biological samples. DsRed is a red fluorescent protein derived from the sea coral Discosoma sp. The antibody can be used in applications such as Western blotting, immunohistochemistry, and immunocytochemistry to visualize the expression and distribution of DsRed-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lysostaphin, by Fujifilm (1 mentions)
Pierce western blot substrate, by Thermo Fisher Scientific (1 mentions)
Living colors anti rcfp polyclonal pan antibody, by Takara Bio (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Alpha actin, by Merck Group (1 mentions)
Cy3 affinipure goat anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Protein a magnetic bead, by Merck Group (1 mentions)
Bioruptor sonicator, by Cosmo Bio (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Mowiol mounting medium, by Merck Group (1 mentions)
Nanozoomer, by Hamamatsu Photonics (1 mentions)
Las 3000 imaging system, by Fujifilm (1 mentions)
Skim milk, by Merck Group (1 mentions)
Strep tactin conjugated to horseradish peroxidase, by IBA Lifesciences (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Nupage 4 12 bis tris polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Anti cat, by Abcam (1 mentions)
8 protocols using dsred polyclonal antibody
1
Fluorescent Protein Detection in S. aureus
2
Confocal Imaging of Zebrafish Embryos
3
Chromatin Immunoprecipitation of RBR1 in Arabidopsis
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Two‐week‐old seedlings of PRORBR1:RBR1:mRFP‐expressing plants growing on ½ MS plates were used. Chromatin was sheared with a Bioruptor sonicator (Cosmo Bio) twice for 15 min with a 50% duty cycle and high power output to obtain 200‐ to 1,000‐bp DNA fragments. Immunoprecipitation was performed using the DsRed polyclonal antibody (Clontech) together with protein A‐magnetic beads (Millipore). The E2FA antibody was described previously (Heyman et al, 2011 ). Negative controls were performed without antibody. DNA was recovered using Magna ChIP spin filters according to the manufacturer's instructions (Millipore). Then, 0.5 or 1 μl of a one‐fifth dilution of ChIP DNA was analyzed by ChIP PCR or quantitative real‐time PCR using gene‐specific primers, respectively (see Appendix Table S2 ). Two biological and three technical replicates were performed for ChIP–quantitative PCR using MCM5 and ORC3 as positive controls and heterochromatic region primers as a negative control (see Appendix Table S2 ).
4
Whole-mount immunostaining in zebrafish larvae
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Whole-mount immunostaining was performed as described (Boezio et al., 2020 (link)). Larvae were fixed in 4% paraformaldehyde for 2 h at room temperature after stopping the heart with 0.4% tricaine. The fixative was substituted with PBS containing 0.1% Tween-20 and the yolks were manually removed using forceps. The blocking step preceding primary antibody incubation was performed in PBS with 1% bovine serum albumin, 1% DMSO and 0.5% Triton X-100 supplemented with 5% goat serum. Primary antibody incubations were performed overnight at 4°C at the following dilutions: anti-GFP (1:400, chicken; cat. no GFP-1020, AvesLabs), anti-Cav1 (1:50, mouse; cat. no 610406, BD Biosciences) and DsRed polyclonal antibody (1:200, rabbit; cat. no. 632496, Takara). All secondary antibodies tagged with Alexa Fluor 568, Alexa Fluor 488 and Alexa Fluor 647 (Thermo Fisher Scientific) were incubated overnight at 4°C at a 1:500 dilution. Larvae were incubated with 1 µg/ml DAPI with the secondary antibody.
5
Immunoprecipitation Assay Protocol
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For immunoprecipitation assays, cells were starved without cAMP pulses for 3 h. Starved cells were lysed in lysis buffer (10 mM NaPi pH 7.2, 100 mM NaCl, 0.5% NP-40, 10% Glycerol, 1 mM NaF, .5 mM Na3VO4, and protease inhibitor) and incubated for 10 min on ice. Lysates were centrifuged for 10 min at 4°C. The supernatants were incubated with GFP-Trap beads for 1 h at 4°C. Beads were washed with lysis buffer. Sample were eluted with SDS loading buffer and subjected to SDS-PAGE followed by mass spectrometry analysis or immunoblotting. Mass spectrometry analysis and immunoblotting were performed as described before (Cai et al., 2010 (link); Yang et al., 2021 (link)). Anti-GFP antibody (Roche 11814460001) and DsRed polyclonal antibody (Takara 632496) were used for immunoblotting.
6
Fluorescent Immunohistochemistry of Nematostella
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Adult Nv-Elav1::mOrange Nematostella were paralyzed in anesthetic solution, then placed in a 4% solution of PFA overnight. Animals were cryoprotected using a gradient of increasing sucrose concentrations (10% to 50%) in PBS over two days. Cryostat sections (20 µm thick) were permeabilized with 0.2% Triton-X and 4% normal goat serum (NGS) at room temperature for 1 hr, followed by incubation with DsRed Polyclonal Antibody (Takara Bio Cat# 632496, RRID:AB_10013483 ) overnight in PBST (0.2%) and NGS (4%) at 4°C. Tissue was rinsed three times with PBST before secondary was applied (Goat anti-rabbit 647, Abcam in PBST + NGS) for 2 hr at room temperature. Tissue was rinsed with PBS and mounted with Vectashield containing DAPI (Novus Biologicals).
7
Mapping DREADD-Infected ReRh Neurons
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To evaluate the viral infection in ReRh, brain sections from DREADD-injected rats (experiment 2) were rinsed 3 × 10 min in PBS before being mounted using Mowiol mounting medium (Sigma-Aldrich). Direct fluorescence of the AAV-encoded mCherry was visualized using a Nanozoomer (Model S60, Hamamatsu). The fibers and terminal sites of ReRh-infected cells were observed after an amplification of mCherry signals and a counterstaining with calretinin performed on sections of frontal and hippocampal regions. The immunostaining protocol was similar to the one used for c-Fos immunohistochemistry. A DsRed polyclonal antibody (1/1000, Takara) was used to amplify the mCherry fluorescence and, for counterstaining, a mouse calretinin antibody (1:8000, rabbit, Swant) was used as the primary antibody, and an Alexa A488 anti-mouse antibody (1:1000; Invitrogen) as the secondary one.
8
Quantitative Western Blot Analysis
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Mycoplasma cell lysates were quantified using the Pierce ™ BCA Protein Assay Kit. Cell extracts were separated by electrophoresis using NuPage™ 4-12% Bis-Tris polyacrylamide gels (Invitrogen) and proteins transferred onto nitrocellulose membranes using an iBlot™ dry system (Invitrogen). Membranes were blocked in a PBS buffer containing 0.1% Tween 20 supplemented with 5% skim milk (Sigma) or 3% BSA in the case of Strep-tag detection. For immunodetection, membranes were probed with polyclonal anti-CAT (abcam, 1:2,000), monoclonal anti-FLAG M2 (Sigma, 1:5,000), Strep-Tactin conjugated to horseradish peroxidase (IBA lifesciences, 1:10,000), DsRed polyclonal antibody (Takara, 1:2,000), anti-polyclonal anti-ssrAmk tag (1:2,000) or polyclonal antibodies specific to mycoplasma proteins (kind gift of Dr. Herrmann, Heidelberg University). These include anti-Lon (1:3,000) and anti-FtsH (1:3,000), which were also used as loading controls together with anti-CAT (abcam, 1:2,000) antibody. Anti-mouse IgG (1:10,000) or anti-rabbit IgG (1:5000) conjugated to horseradish peroxidase (Sigma) were used as a secondary antibody. Blots were developed with the Supersignal™ West Femto or West Pico Chemiluminescent Substrate detection Kit (Thermo Scientific) and signals detected in a LAS-3000 Imaging System (Fujifilm).
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