F1804 5mg
Manufactured by Merck Group
Sourced in United Kingdom, United States
F1804-5MG is a laboratory product from Merck Group. It is a chemical compound used in various research and analytical applications. The core function of this product is to serve as a reference material or standard for analytical procedures. No further details about its intended use or applications are provided.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Am4300, by Thermo Fisher Scientific (1 mentions)
Ab2900, by Abcam (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 anti rabbit, by Thermo Fisher Scientific (1 mentions)
Spinning disc confocal microscope, by Nikon (1 mentions)
D110087, by Sangon (1 mentions)
G6532, by Merck Group (1 mentions)
A600669 0250, by Sangon (1 mentions)
D110058, by Sangon (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Bes5101, by BersinBio (1 mentions)
5 protocols using f1804 5mg
1
Immunoblotting with Anti-FLAG and GAPDH
2
Investigating TIMP-3 Binding and Internalization
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HTB94 cells (104 cells) were plated on gelatin-coated coverslips in 12-well plates. Wells were washed in serum-free DMEM and incubated for 2 h at 37 °C with TIMP-3, TIMP-3 K26A/K45A, or TIMP-3 K42A/K110A (40 nm ) in DMEM with 0.1% FCS. Control wells were incubated without TIMP-3, and LRP dependence was evaluated by incubating wells with RAP (500 nm ) for 1 h before the addition of TIMP-3. Wells were washed three times in PBS after each of the steps hereafter. Cells were fixed with 3% paraformaldehyde plus PBS (10 min, 25 °C), blocked with 5% goat serum plus 3% BSA plus PBS (1 h, 25 °C), and permeabilized in 0.1% Triton X-100 plus PBS (15 min, 25 °C). Cells were stained with mouse anti-FLAG M2 (5 μg/ml F1804-5MG, Sigma-Aldrich) and rabbit anti-EEA1 and 0.5 μg/ml ab2900 (AbCam, Cambridge, UK) antibodies in block (3 h, 25 °C). Bound primary antibodies were detected with anti-mouse DyLight 679 (ThermoFisher, Waltham, MA) and anti-rabbit Alexa Fluor 568 (Molecular Probes, Inc., Eugene, OR) in block (1 h, 25 °C). Nuclei were visualized (1 h, 25 °C) with DAPI (ThermoFisher), and actin was visualized with phalloidin Alexa Fluor 488 (Molecular Probes). Cells were mounted in ProLong Diamond antifade mountant (ThermoFisher) and analyzed on a PerkinElmer Life Sciences spinning disc confocal microscope equipped with a Nikon TE-2000 CCD camera (×40 objective lens).
3
Western Blot Protein Analysis Protocol
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Bacterial samples were resuspended in 1× protein loading buffer and boiled at 98 °C for 8 min. 0.05 OD of proteins samples were loaded each lane. Proteins were transferred onto PVDF membranes (#10600023, Cytiva) for 45 min at 150 V in transfer buffer. Membranes were blocked for 1 h at room temperature in 1× TBST buffer with 5% (w/v) skim milk (#A600669-0250, Sangon), washed with TBST twice and incubated with monoclonal α-FLAG (Sigma-Aldrich #F1804-5MG; 1:10,000), α-SipC (1: 3,000) or α-GroEL (Sigma-Aldrich #G6532; 1:10,000) antibodies for 1 h at room temperature. After three TBST washes, membranes were incubated with secondary α-mouse or α-rabbit HRP-linked antibodies (Sangon #D110087 or #D110058; 1:10,000) for 1 h at room temperature. Chemiluminescence was developed using the high sensitive ECL luminescence reagent (#C500044-0100, Sangon), and then visualized on ChemiScope 6000SE and quantified using ImageJ Software.
4
Evaluating Recombinant ORFV Viruses' Replication and Gene Expression
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Replication kinetics of ORFVΔ121cH6/1 or ORFVΔ121cH8/1 recombinant viruses were assessed through single- and multi-step growth curves in OFTu and STu cells and compared to the parental OV-IA82, as previously described (5 (link)). Briefly, for the multistep and single-step growth curves, the cells were infected at 0.1 MOI and 10 MOI, respectively, and subsequently harvested at 6, 12, 24, 48, and 72 hours post-infection (hpi). The controls were mock-infected OFTu and STu collected at 0 hpi. Viral titers were determined at each time point by limiting dilution and expressed as tissue culture infectious dose 50 (TCID50) per milliliter.
To evaluate the expression of heterologous genes by the recombinant viruses, OFTu cells were infected with either ORFVΔ121cH6/1 or ORFVΔ121cH8/1, and evaluated by indirect IFA, flow cytometry, and Western blot, as previously described (49 (link)). Anti-FLAG tag epitope monoclonal mouse antibody (Sigma-Aldrich) was used for the detection of the fusion cH6/1- or cH8/1-flag proteins in all assays.
The stability of both cH6/1 and cH8/1s genes inserted into the ORFV121 locus was evaluated during 10 serial passages of the respective recombinant viruses in OFTu cells by assessing expression through IFA by using a monoclonal anti-FLAG M2 mouse antibody (1:500, F1804-5MG, Sigma-Aldrich), as previously described (49 (link)).
To evaluate the expression of heterologous genes by the recombinant viruses, OFTu cells were infected with either ORFVΔ121cH6/1 or ORFVΔ121cH8/1, and evaluated by indirect IFA, flow cytometry, and Western blot, as previously described (49 (link)). Anti-FLAG tag epitope monoclonal mouse antibody (Sigma-Aldrich) was used for the detection of the fusion cH6/1- or cH8/1-flag proteins in all assays.
The stability of both cH6/1 and cH8/1s genes inserted into the ORFV121 locus was evaluated during 10 serial passages of the respective recombinant viruses in OFTu cells by assessing expression through IFA by using a monoclonal anti-FLAG M2 mouse antibody (1:500, F1804-5MG, Sigma-Aldrich), as previously described (49 (link)).
5
Detecting AC092894.1 via RIP
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