Immnunolabeling was carried out by reacting the Cy3-Alexa647 heterodimer with secondary antibodies (anti-mouse IgG produced in goat, F5262, Sigma Aldrich; anti-rabbit IgG produced in goat, A0545, Sigma Aldrich) in sodium borate buffer (0.1 M, pH = 8.5, 71999, Sigma Aldrich). The final concentrations of the reagents were roughly 200 μM and 4 μM for the heterodimer and secondary antibody, respectively. The amine groups of the secondary antibody react with the remaining NHS-ester functional group of the heterodimer. The reaction was kept on for more than 12 hours at 4 °C, after which the product was purified by a size-exclusive desalting column (PD-10, GE Healthcare) with phosphate buffered saline (PBS, 1×). We picked the fraction that shows three absorption peaks (protein, Cy3 and Alexa647) simultaneously in the UV/VIS absorption spectrum. By measuring the absorption and emission spectra, we confirmed that the optical properties of the heterodimer were well preserved after labeling (see Supplementary Fig. S5 online). We tested the activity of our heterodimer-labeled antibody by confirming the co-localization with commercially available FITC-labeled secondary antibody (F5262, Sigma Aldrich) (see Supplementary Fig. S6 online).
F5262
Manufactured by Merck Group
Sourced in United Kingdom
The F5262 is a laboratory equipment product. It serves as a core function in laboratory environments. No further details are available.
Automatically generated - may contain errors
Lab products found in correlation
Ab91262, by Abcam (1 mentions)
Ab28340, by Abcam (1 mentions)
Ab150187, by Abcam (1 mentions)
Ab77192, by Abcam (1 mentions)
Ab32886, by Abcam (1 mentions)
P0448, by Agilent Technologies (1 mentions)
A11034, by Thermo Fisher Scientific (1 mentions)
G2654, by Merck Group (1 mentions)
Radiance 2100, by Bio-Rad (1 mentions)
A0545, by Merck Group (1 mentions)
Pd 10, by GE Healthcare (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
H9658, by Merck Group (1 mentions)
4 protocols using f5262
1
Heterodimer-Labeled Antibody Immunolabeling
2
Multimarker Immunohistochemistry of Pancreatic Islets
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Pancreases or isolated islets from Wistar rats, GK rats and human subjects were fixed in 4% formalin, paraffin embedded and cut into 5‐μm sections. For immunofluorescence, sections were dewaxed and rehydrated as described above. All samples were subjected to antigen retrieval by heating in 0.01 M sodium citrate solution before immunolabelling. Sections were incubated overnight using antibodies targeting PYY (ab22663), insulin (in house), glucagon (G‐2654, Sigma, Gillingham, UK), somatostatin (sc‐25262, Santa Cruz, Insight Biotechnologies, Wembley, UK), PP (ab77192), NPY1R (ab91262, Abcam, Cambridge, UK), NPY2R (ab31894, Abcam), NPY4R (HPA027863, Sigma), NPY5R (ab32886, Abcam) or DPP‐IV (ab28340, Abcam). The tyramide amplification system was used as a secondary antibody system to improve visualization of PYY in human samples, all NPYR proteins and DPP‐IV (ThermoFisher, Loughborough, UK). All other proteins were visualized using fluorescently‐ and HRP‐labelled secondary antibodies for immunolocalization as follows; anti‐rabbit 488 (A11034, Life Technologies, Loughborough, UK), anti‐mouse TRITC (F5262, Sigma), anti‐guinea pig 648 (ab150187, Abcam), anti‐rabbit HRP (P0448, Dako, Cheadle, UK). Images were visualized using a BioRad (Radiance 2100) confocal microscope.
3
In Vitro Norovirus Protein Expression
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Example 9
To determine in vitro protein expression of the constructs, HeLa cells are transiently transfected with mRNA encoding Norovirus antigens and stained using suitable customized anti Norovirus-protein antibodies (raised in mouse) and a FITC-coupled secondary antibody (F5262 from Sigma).
Hela cells are seeded in a 6-well plate at a density of 400000 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep), 24 h prior to transfection. Hela cells are transfected with 1 and 2 μg unformulated mRNA using Lipofectamine 2000 (Invitrogen). The mRNA constructs are used in the experiment, including a negative control encoding an irrelevant protein. 24 hours post transfection. Hela cells are stained with suitable anti Norovirus-protein antibodies (raised in mouse; 1:500) and anti-mouse FITC labelled secondary antibody (1:500) and subsequently analyzed by flow cytometry (FACS) on a BD FACS Canto II using the FACS Diva software. Quantitative analysis of the fluorescent FITC signal is performed using the FlowJo software package (Tree Star, Inc.).
4
Trafficking Assay for F508del-CFTR
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The trafficking assay was performed as previously described by Carlile et al.21 (link). In brief, 3HA-tagged F508del-CFTR expressing baby hamster kidney (BHK) cells (cells earlier described by Carlile et. al. 2007)21 (link) were seeded in 96-well plates (Corning half area, black-sided, clear bottom) at 15,000 cells per well. After 24 h incubation at 37 °C, the cells were treated with 10 μM of compound for 24 h (final DMSO concentration 1% v/v). The cells were fixated with 4% paraformaldehyde, washed with PBS, and then blocked with fetal bovine serum (5% in PBS). Mouse monoclonal anti-HA antibody (H9658 Sigma, 1:150 dilution in PBS) was incubated overnight, and, after three washes with PBS, the background fluorescence was measured in a plate reader (488 nm excitation, 510 nm emission). The secondary antibody anti-mouse IgG conjugated with FITC (F5262 Sigma, 1:100 dilution in PBS) was incubated for 1 h, the cells were washed three times with PBS and analyzed in the plate reader again. Background fluorescence was subtracted from the signal, after which the signal was normalized to the DMSO control and wt-CFTR expressing cells.
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