Irdye800

Manufactured by Thermo Fisher Scientific

IRDye800 is a near-infrared fluorescent dye used in various biomedical applications. It has a peak excitation wavelength of 774 nm and a peak emission wavelength of 789 nm. The dye is designed to provide high fluorescence intensity and photostability for sensitive detection and imaging.

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6 protocols using irdye800

Immunofluorescence and Western Blot Protocols

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For immunofluorescence the following primary antibodies were used: GFP (Santa Cruz, sc-9996) and TGN46 (Thermo, pa 1-1069). Secondary antibodies used are the following: Alexa 488 (Invitrogen, a21202 and a21206), Alexa 594 (Invitrogen, a21442) and Alexa 647 (Invitrogen, a281883). For Western Blot the following primary antibodies were used: GFP (Santa Cruz, sc-9996), actin (Abcam, ab6276), Ykt6 (Abcam, ab236583), LC3B (Cell Signaling, #2775), p62
(Sigma, p0067), STX17 (Sigma, hpa001204), SNAP29 (Abcam, ab138500) Na+/K+ ATPase (Sigma, 06-172-I), alpha/beta-Tubulin (Cell Signaling, 2148S), and STX7 (Bethyl, A304-512A).
Secondary antibodies used are the following: IRDye680 (Fisher, 925-68070) and IRDye800
(Fisher, 925-32211). Secondary antibodies used are the following: IRDye680 (Fisher, 925-68070) and IRDye800 (Fisher, 925-32211).

McGrath K., Dergai M., Agarwal S., Chung D., Rossum D.B.v., Shevade A., Kuchin S., Zaichick S., Savas J.N., Fasshauer D., & Caraveo G. (2020). A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy.

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Western Blot Analysis of HMGB1 and TLR4

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Thirty micrograms of renal cortex was homogenized, and the whole homogenate was used for Western blot analysis as previously described [27 (link)]. Briefly, samples were resolved on 4–20% Tris-glycine-SDS gels (Bio-Rad, Hercules, CA) and proteins were transferred to PVDF membranes (Millipore Sigma, Burlington, MA). Protein expression was determined using two-color immunoblots using primary antibodies to HMGB1 (cat# MA1–20338, 1:1000 dilution; Thermo Fisher Scientific, Lafayette, CO), and TLR4 (cat# 217274, 1:300 dilution; Abcam, Cambridge, MA). Specific protein bands were detected using an Odyssey infrared imager (LI-COR Biosciences, Lincoln, NE) with AlexaFluor 680 and IRDye800 (Molecular Probes, Eugene, OR) conjugated secondary antibodies. Protein concentrations were determined by standard BCA reagent (Thermo Scientific) using BSA as the standard. Protein loading was normalized to glyceraldehyde 3-phosphate dehydrogenase (Cell Signaling Technologies, Danvers, Massachusetts).

Mohamed R., Rafikova O., O’Connor P.M, & Sullivan J.C. (2020). Greater high-mobility group box 1 in male compared with female spontaneously hypertensive rats worsens renal ischemia–reperfusion injury. Clinical science (London, England : 1979), 134(13), 1751-1762.

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Western Blot Analysis Protocol

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Western blots were performed according to our standard protocol (26 (link)). Briefly, cell lysates containing 25 or 50 µg protein were separated by SDS-polyacrylamide gel electrophoresis, transferred onto a PVDF membrane (Millipore) and subjected to western blot analysis with the recommended dilution of primary and appropriate secondary antibody conjugated to IR Dye 680 or IR Dye 800 (Molecular Probes). The membranes were scanned by Odyssey Infrared Imaging System (LI-COR Biosciences) to locate the respective bands. β-actin or GAPDH was used as a loading control.

Vasudevan A., Yu Y., Banerjee S., Woods J., Farhana L., Rajendra S.G., Patel A., Dyson G., Levi E., Maddipati K.R., Majumdar A.P, & Nangia-Makker P. (2014). Omega-3 fatty acid is a potential preventive agent for recurrent colon cancer. Cancer prevention research (Philadelphia, Pa.), 7(11), 1138-1148.

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Analyzing Kidney Phospho-c-Jun Levels

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Frozen kidney samples were homogenized in 0.5 ml lysis buffer and processed for Western blotting as previously described (Ma et al., 2014 (link)). Blots were probed for phospho-c-Jun and α-tubulin as the loading control using goat anti-rabbit Alexa Fluor 680 or donkey anti-mouse IRDye 800 secondary antibodies (Molecular Probes) and detected using the Odyssey Infrared Image Detecting System (LICOR). Densitometry analysis used ImageJ software, NIH.

Yang F., Ozols E., Ma F.Y., Leong K.G., Tesch G.H., Jiang X, & Nikolic-Paterson D.J. (2021). c-Jun Amino Terminal Kinase Signaling Promotes Aristolochic Acid-Induced Acute Kidney Injury. Frontiers in Physiology, 12, 599114.

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ADAM23 Protein Expression Analysis

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Western blot analysis was performed using the previously described protocol [20 (link)]. The primary human ADAM23 antibody (Santa Cruz) is a rabbit polyclonal antibody raised against amino acids 351–415 (etwtekdqidittnpvqmlhefskyrqrikqhadavhlisrvtfhykrsslsyfggvcsrtrgvgand) within an extracellular domain of human ADAM23 (NP_003803). The primary chicken ADAM23 antibody (generated by a custom service from Eurogentec) is a rabbit polyclonal antibody against amino acids asmqlqdhetesssew and cirdtgnkkdegpkgb of chicken ADAM23 (NP-001138702.1) [25 (link)].
Note that the epitope sequences of the antibodies against human and chicken ADAM23 were not compatible, so there was no cross-reaction between the species in the Western blot detection. GAPDH (Abcam) and the secondary antibodies of Alexa Fluor 680 from goat anti-rabbit or anti-mouse, and of goat anti-mouse IRDye 800 (all from Invitrogen) were used. GAPDH was used as a loading control for the Western blot. Protein visualization and quantification were performed with an Odyssey Infrared Imaging System (LI-COR Biosciences GmbH) according to the manufacturer’s instructions.

Markus-Koch A., Schmitt O., Seemann S., Lukas J., Koczan D., Ernst M., Fuellen G., Wree A., Rolfs A, & Luo J. (2017). ADAM23 promotes neuronal differentiation of human neural progenitor cells. Cellular & Molecular Biology Letters, 22, 16.

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Western Blot Analysis of Phosphorylated Proteins

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Sorted cells were boiled in Laemmli sample buffer for 5 min prior to separation on 10% SDS-polyacrylamide gels. Proteins were transferred to PVDF membrane (Millipore, Etten Leur, The Netherlands) by semidry electroblotting. Membranes were blocked in Odyssey blocking buffer (Westburg, Leusden, the Netherlands) prior to incubation with antibodies. Binding of antibodies was detected by incubating with Alexa680 or IRDye800 labeled secondary antibodies (Invitrogen, Breda, the Netherlands) and scanning of the membrane on an Odyssey infrared scanner (Li-Cor Biosciences, Lincoln, NE, USA). Signal intensities were quantified using Odyssey 2.1 analysis software and calculated relative to the highest intensity. Antibody against against phosporylated ERK (pT202,pY204) was obtained from Cell Signaling technologies (Leiden, the Netherlands), antibody against tyrosine phosphorylated STAT5 was obtained from Becton Dickinson (Breda, the Netherlands) and antibodies against ERK (K23) and STAT5 (C17) were obtained from Santa Cruz (Santa Cruz Biotech, Santa Cruz, CA, USA). Anti myc antibody was obtained from Roche (Almere, the Netherlands).

Wierenga A.T., Vellenga E, & Schuringa J.J. (2014). Convergence of Hypoxia and TGFβ Pathways on Cell Cycle Regulation in Human Hematopoietic Stem/Progenitor Cells. PLoS ONE, 9(3), e93494.

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