I will be using Biorad Stain Free gels with the plan being to image the fluorescence and total protein on the gel, before Western blotting for the proteins Strep tag. The cells were then intracellularly stained with 5 µg/ml Alexa Fluor ® 594 anti-Nuclear Pore Complex Proteins antibody (clone MAb414, red in blocking buffer three hours at room temperature. also are there other programs out there? Can you help by adding an answer? Then the section was stained with 10 µg/ml of purified anti-mouse B220 (clone RA3-6B2), and anti-mouse CD3ε (clone 145-2C11) Alexa Fluor® 647 (green) overnight at 4°C, followed by 2.5 µg/mL of Alexa Fluor® 594 anti-rat IgG (clone Poly4054) (red) for two hours at room temperature. In addition, is used in a range of SRM applications including dSTORM, SIM, and STED and in two-photon excitation (TPE) microscopy. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company. This is a table of some characteristics of fluorochromes useful for flow cytometry or fluorescence microscopy. The NHS ester (or succinimidyl ester) of Alexa Fluor® 594 is the most popular tool for conjugating this dye to a protein or antibody. What is the recipe for 0.01% Tween-20 in PBS? I will very appreciate that if you can share with me how is it going with these Alexa Fluor conjugated secondary antibodies on Typhoon? No. Scale bar for histological images = 20 µm. Can anyone recommend a good free FACS analysing software? Does anyone do this in X. laevis for whole-mount staining? Alexa Fluor 594 dye molecules can be attached to proteins at high molar ratios without significant self-quenching, enabling brighter conjugates and more sensitive detection. For stable signal generation in imaging and flow cytometry, Alexa Fluor 594 dye is pH-insensitive over a wide molar range. All rights reserved. Microtubules of fixed bovine pulmonary artery endothelial cells were visualized with, Chromatography Columns, Resins, & Spin Filters, Spectroscopy, Elemental & Isotope Analysis, Preclinical to Companion Diagnostic Development, Super-resolution microscopy (SRM) applications, Streptavidin conjugates for signal amplification, Tyramide labeling kits for ultimate sensitivity, Annexin V conjugates for apoptosis imaging, Labeled nucleotides for hybridization probes, See all Alexa Fluor 594 Primary Antibodies, See all Alexa Fluor 594 Secondary Antibodies, Alexa Fluor 350 goat anti–mouse IgG antibody, Zenon Alexa Fluor 594 Mouse IgG1 Labeling Kit, Zenon Alexa Fluor 594 Mouse IgG2a Labeling Kit, Zenon Alexa Fluor 594 Mouse IgG2b Labeling Kit, Zenon Alexa Fluor 594 Human IgG Labeling Kit, Zenon Alexa Fluor 594 Rabbit IgG Labeling Kit, APEX Alexa Fluor 594 Antibody Labeling Kit, Alexa Fluor 594 Microscale Antibody/Protein Labeling Kit, Alexa Fluor 594 Antibody/Protein Labeling Kit, Cell Analysis Learning Center—Imaging information, 5 steps to publication-quality fixed-cell imaging. Okinawa Institute of Science and Technology. The average molecular weight of Streptavidin-Alexa Fluor® 594 is 60 kD and Streptavidin alone is 52 kD. Does anybody have experience with using BSA instead of milk for blocking and using TBS-T instead of PBS-T? What is appropriate incubation time for primary antibody in western blot analysis? Do not freeze. (TIF). Just on a side note, uvGFP is typically excited at 395 nm, and apparently the UV tray we have with our Biorad EZ gel-doc does give a UV source that covers 395 mm. Thermo Fisher Scientific. Microtubules of fixed bovine pulmonary artery endothelial cells were visualized with mouse monoclonal anti–α-tubulin antibody, followed by an Alexa Fluor 350 goat anti–mouse IgG antibody. I used PBMC activated with pokeweed and stained with anti IFNg (intracellular -657) and CD4-FITC. Find out what wavelengths of detection the Typhoon is configured for. Sort the right letters to the bars gets much more complex when the number of bars increases. The secondary antibody (orange) was ab150156 Alexa Fluor® 594 donkey anti-rat IgG (H+L) used at 1µg/ml for 1h. some of the percentages of population rose from ca 0.5% (cell quest) to around 20% (WinMDI) any ideas? I'm using these detergents for whole-mount antibody staining in X. laevis. HeLa cells were fixed with 100% methanol for 15 minutes at -20°C followed by blocking with 5% FBS for 30 minutes. Streptavidin-Alexa Fluor® 594 is useful as a second step reagent for indirect immunofluorescent staining, when used in conjunction with biotinylated primary antibodies. From various protocols I've come across, I understand that I can run the protein on a normal gel with my usual SDS sample buffer as GFP is stable enough to still fluorese, but I should not boil (which I would not do with a membrane protein anyway) or heat the samples in any way. Dye 5' Int 3' Ex. We offer Alexa Fluor 594 dye conjugated to a variety of antibodies, peptides, proteins, tracers, and amplification substrates optimized for cellular labeling and detection. In addition, reactive dye forms and protein labeling kits are available to allow you to generate your own antibody conjugates or probes. Alexa Fluor® 594 conjugates are brighter than red-fluorescing conjugates, and they provide more color separation from green-fluorescing dyes than DyLight 549, Cy3, and TRITC conjugates. I need to detect two different proteins by western blot (55 and 38 MW), but I don't want to strip my membrane twice. Nuclei were counterstained with DAPI (blue). Search Used for stable signal generation in imaging and flow cytometry, Alexa Fluor® 594 dye is water soluble and pH-insensitive from pH 4 to pH 10. I assume that I should therefore just leave them at RT for an hour or so? The Alexa Fluor dyes—a series of superior fluorescent dyes that span the visible … Next, the F-actin was labeled with Alexa Fluor 594 phalloidin. AFDye™ 594 NHS Ester (Alexa Fluor 594 NHS Ester) is the most popular tool for modifying proteins or antibodies through the primary amines (Lys), amine-modified oligonucleotides, and other amine-containing biomolecules with AFDye™ 594 label. Alexa Fluor® 6471 RPA-T4 660/20 184 PE-Cy™7 RPA-T4 780/60 122 PerCP-Cy™5.52 RPA-T4 695/40 99 Alexa Fluor® 4883 RPA-T4 530/30 68 BD Horizon™ V4505 RPA-T4 450/50 65 Alexa Fluor… Negative control immunofluorescent staining in BCA with mouse IgG1 isotype control and Alexa Fluor 488 or Alexa Fluor 594 labeled secondary antibodies. Any further comments would be incredibly helpful and very much appreciated! Do you think I would be able to image the gel using that to excite the uvGFP or would I need to find a dedicated fluorescence imager? No. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. Em. I have several hundreds of statistical comparisons here and have to find a computer program that can generate the letters from the data for me. Apparatus used is BioRad Mini-Transblot (tank/wet transfer method). Western blotting - using BSA or milk? Confocal immunofluorescent analysis of THP-1 cells differentiated with TPA (12-O-Tetradecanoylphorbol-13-Acetate) #4174 (80 nM, 24 hr; left, positive) and SH-SY5Y cells (right, negative) using Iba1/AIF-1 (E4O4W) XP ® Rabbit mAb (Alexa Fluor ® 594 Conjugate) (red). Properties and structures A range of Alexa dyes is available, with emission wavelengths spanning the visible spectrum and extending into the infrared region of the spectrum. And TBS-T or PBS-T? Alexa Fluor 594 dye Invitrogen Alexa Fluor 594 dye is a bright, red-fluorescent dye that can be excited using the 561 nm or 594 nm laser lines. (A) Cultured RPE1 cells were infected with C. trachomatis LGV2 for the indicated timepoints prior to fixation with 1% paraformaldehyde. Primary antibodies, Incubation time, western blotting, ELISA, SDS-PAGE. Flow cytometric analysis of 293 cells, wild-type (blue) or stably transfected with a construct expressing FLAG-Tag (green), using DYKDDDDK Tag (D6W5B) Rabbit mAb (Binds to same epitope as Sigma's Anti-FLAG ® M2 Antibody) (Alexa Fluor ® 594 Conjugate) (solid lines) compared to concentration-matched Rabbit (DA1E) mAb IgG XP ® Isotype Control (Alexa Fluor ® 594 Conjugate) #8760 (dashed lines). The superimposed and pseudocolored images were acquired sequentially using bandpass filter sets appropriate for DAPI, Texas Red dye, and fluorescein, respectively. Alexa Fluor® - Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) preadsorbed (ab150088) Protocols. The data of the statistical test is available in the following format: I want to mark significant differences between two bars with different letters (like bar1:a and bar2:b). Transfer buffer used was Bjerrum Schafer-Nielsen buffer (48 mM Tris, 39 mM glycine, pH 9.2, containing 20% methanol) containing 0.1% SDS. For stable signal generation in imaging and flow cytometry, Alexa Fluor 594 dye is pH-insensitive over a wide molar range. Then the cells were intracellularly stained with (A) Alexa Fluor® 594 Mouse IgG2b, κ Isotype Ctrl Antibody (Negative, Cat. Alexa Fluor® 594 anti-mouse CD8a Antibody - CD8, also known as Lyt-2, Ly-2, or T8, consists of disulfide-linked α and β chains that form the α(CD8a)/β(CD8b) heterodimer and α/α homodimer. Initial transfer performed overnight (16hr) at 30V failed because small and mid sized proteins (smaller than 100kDa) transferred right through the membrane (as evident from Ponceau staining of the blot and Coomassie staining of the gel). DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. I'm ordering reagents for Western blotting, the experiment will be started, I think, maybe 1 month later. How to denote letters to mark significant differences in a bar chart plot. Add your answer. The antibody was purified by affinity chromatography and conjugated with Alexa Fluor® 594 under optimal conditions. I was talking to a postdoc and they said I should wash with 0.01% Tween-20 in PBS (PBS-Tween 20), and antibody stain with 0.01% Triton X-100 in PBS (PBS-TritonX) since Tween-20 is a less harsh detergent than Triton X-100. AF594 phalloidin conjugate is chemically equivalent to Alexa Fluor® 594 phalloidin (Alexa Fluor is the trademark of ThermoFisher) Alexa Fluor® 594-conjugated antibodies absorb light maximally around 591 nm and fluoresce with a peak around 614 nm. What among all these would work best for cohesive end ligation and successful cloning? The secondary antibody (orange) was ab150112 Alexa Fluor® 594 donkey anti-mouse IgG (H+L) used at 2µg/ml for 1h. Table of Fluorochromes. Not for use in diagnostic procedures. What power conditions (volts or mA) should I use in Western blotting for transferring a 25kDa protein onto a 0.22 micron nitrocellulose membrane? Alexa Fluor® - Donkey Anti-Rat IgG H&L (Alexa Fluor® 594) preadsorbed (ab150156) Protocols. AFDye™ 594 Azide (Alexa Fluor® 594 equivalent) is a bright, red-fluorescent azide-activated probe that reacts with terminal alkynes via a copper-catalyzed click reaction (CuAAC). Can I use the antibodies simultaneously? The Alexa®, or Alexa Fluor®, dyes were introduced by Molecular Probes, Inc. (now part of Life Technologies, Inc.). NHS esters can be used to label to the primary amines (R-NH 2) of proteins, amine-modified oligonucleotides, and other amine-containing molecules. Cite. They are brighter, more photostable and more hydrophilic than Texas Red conjugates. It can be used with the 561 nm and 594 nm laser lines. My lab also has kinds of Alexa Fluor conjugated secondary antibodies, like 488, 546, probably from Invitrogen (I'll check it later). What factors decide the selection of ligation reaction conditions out of these? *** If any one can help me to obtain a good reference material that guide to Interpretation and analysis of biological research data would be much grateful. Fixed cells were stained with an anti-Chlamydia primary antibody and an Alexa Fluor 488-conjugated secondary antibody. In-gel GFP fluorescence with SDS-PAGE protocol? I'm also going to do fluorescent Western detection with Typhoon 9410 and struggling with the secondary antibodies to be chosen. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. Direct replacement for Invitrogen Texas Red dye with bright signal and excellent photostability, especially in fixed cells; commonly used with Invitrogen Alexa Fluor 350, 488, and 647 dyes for multiplexing. Confocal immunofluorescent analysis of COS-7 cells, transiently transfected with a construct expressing Myc-tagged chorionic somatomammotropin hormone-like 1 (CSL), using Myc-Tag (9B11) Mouse mAb (Alexa Fluor ® 594 Conjugate) (red) and β-Catenin (L54E2) Mouse mAb (Alexa Fluor ® 488 Conjugate) #2849 (green). For Research Use Only. Can I detect two different proteins in a western blot using two antibodies? Alexa Fluor® fluorescent dyes are widely recognized as superior fluorescent dyes available for conjugation. The issue is that before buying expensive secondary antibodies, we'd like to know wether or not would be posible using the Alexa Fluor antibodies for WB. IDT product details for Fluorophores modifications. I played around with WinMDI but something weird happend when I comparing the data analysed in cell quest. I am analysing my data with cell quest (which is not my favorite program) I used to use Flowjo (which rocks in my opinion) but have no means to ever get this. The secondary antibody (orange) was ab150088 Alexa Fluor® 594 goat anti-rabbit IgG (H+L) used at 2µg/ml for 1h. Immunofluorescence using; (a) only Alexa Fluor 488 secondary antibody on a population of Paramecium where majority of cells have fragmented parental macronucleus,(b, left panel) C.elegans embryo stained against 5- mCytosine as a negative control for immunofluorescence, (c, right pane... Join ResearchGate to find the people and research you need to help your work. I want to detect phospho-proteins as well as full-proteins. 422801). Answer. Can anyone give me some pointers on how to carry out an SDS-PAGE in-gel fluorescence assay with a uvGFP tagged membrane protein? Learn more about the: Not for resale. Nuclei were counterstained with DAPI (Blue, Cat. The Alexa Fluor family of fluorescent dyes is a series of dyes invented by Molecular Probes, now a part of Thermo Fisher Scientific, and sold under the Invitrogen brand name.Alexa Fluor dyes are frequently used as cell and tissue labels in fluorescence microscopy and cell biology. need your help to find a solution for my problem to indicate significant differences in a bar chart plot. Samples were mounted in ProLong ® Gold Antifade Reagent with DAPI #8961 (blue). © 2008-2021 ResearchGate GmbH. Our neighbour lab has a Typhoon FLA 9500 and they have allowed us to use it, so it would be great in the same gel blot 2 proteins at the same time with same or similar molecular weight. The secondary antibodies were Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (colored green) used at 2 ug/ml and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) (pseudo-colored red) used at 1/1000 dilution for 1hour at room temperature. Similar questions and discussions. The labeling occurs most efficiently at pH 7-9 and forms a stable, covalent amide bond. Thanks a lot. Finally, the cells were incubated with Alexa Fluor 488 wheat germ agglutinin to stain components of endosomal pathways. Invitrogen Alexa Fluor 594 dye is a bright, red-fluorescent dye that can be excited using the 561 nm or 594 nm laser lines. Ligation is carried out at varied temperatures like 16, 22, 25, 37 degrees and for different time like 16 hrs, overnight, 4-6 hours, 2 hrs, 10 mins. They are highly water soluble and remain fluorescent from pH 4 to pH 10. I have added an example plot with letter-coded significant differences to illustrate what I want to do. AFDye 594 C5 Maleimide (Alexa Fluor® 594 C5 Maleimide equivalent) is bright, water-soluble, and pH-insensitive from pH 4 to pH 10 red-fluorescent dye with absorption and emission maxima at 590 and 617 nm, respectively. THANKS. Effect of inhibitor treatment on cell survival and IES retention. 400362) or (B-D) Alexa Fluor® 594 Histone H3 Phospho (Ser10) Antibody (Clone 11D8) overnight at 4°C. The negative control (inset) is a secondary-only assay to demonstrate … Anybody an idea which programme can help me? If I could image the gel on the same gel-doc by just switching between the UV and stain free trays then that would be helpful. Alexa Fluor® 594 - conjugated antibodies absorb light maximally around 591 nm and fluoresce with a peak around 614 nm. The kinetics of C. trachomatis LGV2 entry into cultured RPE1 and HeLa cells. They are the best choice for immunofluorescence detection in the deep-red region of the visible spectrum. They are brighter, more photostable, and more hydrophilic than Texas Red conjugates. If there is no significant differences between two bars they get the same letter (like bar1:a and bar3:a). Select dye-labeling chemistries for conjugation with the following reactive groups: Select an optimized labeling kit for your target antibody or protein: Alexa Fluor dyes are among the most trusted fluorescent dyes available today. What is the best temperature and incubation time for successful ligation? AFDye™ 594 Alkyne reacts with azides via a copper-catalyzed click reaction (CuAAC) to form a stable triazole linker. AFDye™ 594 Alkyne (Alexa Fluor® 594 Alkyne equivalent) is a bright, red-fluorescent alkyne-activated probe routinely used for imaging of azide-containing biomolecules. We offer a broad range of Alexa Fluor 594 products including protein and antibody labeling kits, secondary antibodies, bioconjugates, and more: See all Alexa Fluor 594 Primary Antibodies See all Alexa Fluor 594 Secondary Antibodies. Dear all, we normally use the Alexa Fluor secondary antibodies for IF but we also do a lot of WB and most of the proteins of interest have similar molecular weight so I normally have run several gels to be able to blot most of my proteins (don't like to strip them). Alexa Fluor® - Donkey Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150112) Probes with high fluorescence quantum yield and high photostability allow detection of low-abundance biological structures with great sensitivity. Alternative products to Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) (ab150080) Goat F (ab')2 Anti-Rabbit IgG - (Fab')2 (DyLight® 488), pre-adsorbed (ab102291) It may not have the right components for those wavelengths. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. Concentration 0.5 mg/ml Storage & Handling The antibody solution should be stored undiluted between 2°C and 8°C, and protected from prolonged exposure to light. The isomeric mixture of the amine-reactive monosuccinimidyl ester of Alexa Fluor® 594 dye is available in units of 1 mg, 5 mg, 25 mg or three vials of 100 µg each (A20004, A20104, A37565, A37572), and the 6-isomer of the monosuccinimidyl ester of Alexa Fluor® 610-X dye is available in a 1 mg unit size (A30050). What is the difference between using Triton X-100 and Tween-20? Alternative Category; Alexa Fluor® 488 (NHS Ester) 492: 517 Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye). The Alexa Fluor ones are detected at 488 and 594 nm so I'll give it a try. The electrophoresed gel, membrane and filter pads were equilibrated in transfer buffer around 30mins prior to transfer. Within groups, roughly … With the wide range of Invitrogen Alexa Fluor dyes there is something for every filter and … Alexa Fluor® 594 is a bright, red fluorescent dye that can be excited using the 561 nm or 594 nm laser lines. Does it depend on the antibody and what I am staining for? Some protocols have also said that it essential to run the gel at 4 degrees to avoid degradation, is this actually important? Can someone tell me how to do it? The secondary antibody (orange) was ab150076 Alexa Fluor® 594 donkey anti-rabbit IgG (H+L) used at 2µg/ml for 1h.