Chemidoc xrs imager, by Bio-Rad - Product details

1

Methionine Inhibitor Fluorescent Labeling

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MIF (5 μM) in PBS was treated with 13 (50 μM) along with either 50 μM benzyl isothiocyanate (BITC) or an equivalent volume of DMSO. Reactions were carried out at room temperature for 24 hours. After such time, 50 μL of each reaction was subjected to CuAAC conditions with TMR-N₃ and subsequently treated with 12 μL of 6x SDS loading buffer, followed by SDS-PAGE. The fluorescently labelled protein was then visualized using a ChemiDoc XRS+ imager (Bio-Rad). Gels were then stained with a NOVEX® Colloidal Blue Stain Kit (Invitrogen) and imaged using a ChemiDoc XRS+ imager (Bio-Rad).

Brighty G.J., Botham R.C., Li S., Nelson L., Mortenson D.E., Li G., Morisseau C., Wang H., Hammock B.D., Sharpless K.B, & Kelly J.W. (2020). Using Sulfuramidimidoyl Fluorides that Undergo Sulfur(VI) Fluoride Exchange for Inverse Drug Discovery. Nature chemistry, 12(10), 906-913.

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2

Fluorescent PARP1 Protein Labeling

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PARP1cat (3 μM) in PBS was treated with 2 (100 μM) along with either 10 μM olaparib or an equivalent volume of DMSO. Reactions were carried out at room temperature for 24 hours. After such time, 50 μL of each reaction was subjected to CuAAC conditions with TMR-N₃ and subsequently treated with 12 μL of 6x SDS loading buffer, followed by SDS-PAGE. The fluorescently labelled protein was then visualized using a ChemiDoc XRS+ imager (Bio-Rad). Gels were then stained with a NOVEX® Colloidal Blue Stain Kit (Invitrogen) and imaged using a ChemiDoc XRS+ imager (Bio-Rad).

Brighty G.J., Botham R.C., Li S., Nelson L., Mortenson D.E., Li G., Morisseau C., Wang H., Hammock B.D., Sharpless K.B, & Kelly J.W. (2020). Using Sulfuramidimidoyl Fluorides that Undergo Sulfur(VI) Fluoride Exchange for Inverse Drug Discovery. Nature chemistry, 12(10), 906-913.

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EPHX2 Inhibition and Labeling

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EPHX2 (3 μM) in PBS was treated with 9 (50 μM) along with either 10 μM 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) or an equivalent volume of DMSO. Reactions were carried out at room temperature for 24 hours. After such time, 50 μL of each reaction was subjected to CuAAC conditions with TMR-N₃ and subsequently treated with 12 μL of 6x SDS loading buffer, followed by SDS-PAGE. The fluorescently labelled protein was then visualized using a ChemiDoc XRS+ imager (Bio-Rad). Gels were then stained with a NOVEX® Colloidal Blue Stain Kit (Invitrogen) and imaged using a ChemiDoc XRS+ imager (Bio-Rad).

Brighty G.J., Botham R.C., Li S., Nelson L., Mortenson D.E., Li G., Morisseau C., Wang H., Hammock B.D., Sharpless K.B, & Kelly J.W. (2020). Using Sulfuramidimidoyl Fluorides that Undergo Sulfur(VI) Fluoride Exchange for Inverse Drug Discovery. Nature chemistry, 12(10), 906-913.

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4

Fluorescent PARP1 Protein Labeling

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PARP1cat (3 μM) in PBS was treated with 2 (100 μM) along with either 10 μM olaparib or an equivalent volume of DMSO. Reactions were carried out at room temperature for 24 hours. After such time, 50 μL of each reaction was subjected to CuAAC conditions with TMR-N₃ and subsequently treated with 12 μL of 6x SDS loading buffer, followed by SDS-PAGE. The fluorescently labelled protein was then visualized using a ChemiDoc XRS+ imager (Bio-Rad). Gels were then stained with a NOVEX® Colloidal Blue Stain Kit (Invitrogen) and imaged using a ChemiDoc XRS+ imager (Bio-Rad).

Brighty G.J., Botham R.C., Li S., Nelson L., Mortenson D.E., Li G., Morisseau C., Wang H., Hammock B.D., Sharpless K.B, & Kelly J.W. (2020). Using Sulfuramidimidoyl Fluorides that Undergo Sulfur(VI) Fluoride Exchange for Inverse Drug Discovery. Nature chemistry, 12(10), 906-913.

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5

EPHX2 Inhibition and Labeling

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EPHX2 (3 μM) in PBS was treated with 9 (50 μM) along with either 10 μM 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) or an equivalent volume of DMSO. Reactions were carried out at room temperature for 24 hours. After such time, 50 μL of each reaction was subjected to CuAAC conditions with TMR-N₃ and subsequently treated with 12 μL of 6x SDS loading buffer, followed by SDS-PAGE. The fluorescently labelled protein was then visualized using a ChemiDoc XRS+ imager (Bio-Rad). Gels were then stained with a NOVEX® Colloidal Blue Stain Kit (Invitrogen) and imaged using a ChemiDoc XRS+ imager (Bio-Rad).

Brighty G.J., Botham R.C., Li S., Nelson L., Mortenson D.E., Li G., Morisseau C., Wang H., Hammock B.D., Sharpless K.B, & Kelly J.W. (2020). Using Sulfuramidimidoyl Fluorides that Undergo Sulfur(VI) Fluoride Exchange for Inverse Drug Discovery. Nature chemistry, 12(10), 906-913.

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6

Methionine Inhibitor Fluorescent Labeling

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MIF (5 μM) in PBS was treated with 13 (50 μM) along with either 50 μM benzyl isothiocyanate (BITC) or an equivalent volume of DMSO. Reactions were carried out at room temperature for 24 hours. After such time, 50 μL of each reaction was subjected to CuAAC conditions with TMR-N₃ and subsequently treated with 12 μL of 6x SDS loading buffer, followed by SDS-PAGE. The fluorescently labelled protein was then visualized using a ChemiDoc XRS+ imager (Bio-Rad). Gels were then stained with a NOVEX® Colloidal Blue Stain Kit (Invitrogen) and imaged using a ChemiDoc XRS+ imager (Bio-Rad).

Brighty G.J., Botham R.C., Li S., Nelson L., Mortenson D.E., Li G., Morisseau C., Wang H., Hammock B.D., Sharpless K.B, & Kelly J.W. (2020). Using Sulfuramidimidoyl Fluorides that Undergo Sulfur(VI) Fluoride Exchange for Inverse Drug Discovery. Nature chemistry, 12(10), 906-913.

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7

Western Blot Analysis of Inflammatory Signaling

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Whole cell protein lysates were separated using SDS-PAGE and then transferred to PVDF membrane. Membranes were blocked with 1% (wt/vol) BSA in Tris-buffered saline plus 0.1% Tween-20 (TBS-T) and then incubated in primary antibody with rocking overnight at 4°C. The antibodies for iNOS (BD Transduction Laboratories), NFκB p65 (Santa Cruz Biotechnologies), IKBα (Cell Signaling), phosphop65 Ser536 (Cell Signaling), STAT1 (Cell Signaling), poly ADP ribose polymerase (PARP; Cell Signaling), and phospho-STAT1 (701) (Cell Signaling) were used at 1:1000. Anti β-actin antibody (Sigma Aldrich) was used at 1:4000. After three washes in TBS-T, the membranes were probed with HRP-conjugated secondary antibodies and the proteins were visualized with Bio-Rad Immun-Star Western C chemiluminescence kit. Images were captured and analyzed using a Bio-Rad Chemidoc XRS+ imager (BioRad).

Lawrence D.W., Gullickson G, & Kornbluth J. (2014). E3 ubiquitin ligase NKLAM positively regulates macrophage inducible nitric oxide synthase expression. Immunobiology, 220(1), 83-92.

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8

Western Blot Analysis of Signaling Pathways

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Whole cell protein lysates were separated using SDS-PAGE then transferred to PVDF membrane. Membranes were blocked with 1% (wt/vol) BSA in Tris-buffered saline plus 0.1% Tween-20 (TBS-T) then incubated in primary antibody with rocking overnight at 4 °C. The antibodies for STAT1, pSTAT1 (Tyr701), JAK1, ubiquitin K63 linkage, and pJAK1 were purchased from Cell Signaling. The anti-flag tag (M2) and beta actin antibodies were from Sigma-Aldrich. The anti-NKLAM antibody has been described previously [1 (link)] and the anti-IFNGR1 was purchased from Leinco. After three washes in TBS-T, the blots were probed with HRP-conjugated secondary antibodies and the proteins were visualized with BioRad Immun-Star Western C chemiluminescence kit. Images were captured and analyzed using a BioRad Chemidoc XRS+ imager (BioRad).

Lawrence D.W, & Kornbluth J. (2016). E3 ubiquitin ligase NKLAM ubiquitinates STAT1 and positively regulates STAT1-mediated transcriptional activity. Cellular signalling, 28(12), 1833-1841.

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9

SDS-PAGE and Western Blotting Analysis

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Cells or NMVs were diluted using a buffer and the suspension was loaded in a 12% sulfate-polyacrylamide gel in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then, the bands in SDS-PAGE were transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore Corporation, Billerica, MA), followed by the incubation with primary antibodies (such as anti-integrin β₂, anti-ICAM-1 and anti-GAPDH monoclonal antibody) for 2 h at the room temperature. Then, the blots were incubated with secondary goat anti-mouse IgG-HRP (1:5 000; Santa Cruz Biotechnology) for 2 h. Washed with Tris buffered saline and 0.05% Tween 20 (TBS-T), the blots were treated with the chemiluminescent HRP substrate (Thermo Scientific,) and chemiluminescence was detected using the Bio-Rad ChemiDoc XRS imager (Bio-Rad, Hercules, CA).

Gao J., Dong X., Su Y, & Wang Z. (2021). Human Neutrophil Membrane-derived Nanovesicles as a Drug Delivery Platform for Improved Therapy of Infectious Diseases. Acta biomaterialia, 123, 354-363.

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10

Western Blot Analysis of Protein Targets

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The extraction of cellular proteins and their detection via western blotting were performed as previously described [21 (link), 24 (link)]. Western blotting was performed using the following primary antibodies: mouse anti-AR, mouse anti-beclin1, goat anti-LC3β, mouse anti-LAMP1, and rabbit anti-IKKγ antibodies (1:500, Santa Cruz Biotechnology); mouse anti-TLR4, rabbit anti-iNOS, rabbit anti-IKKα, and rabbit anti-4-HNE (1:1000, Abcam); rabbit anti-IKKβ and rabbit anti-phospho-IκBα antibodies (1:1000, Epitomics); rabbit anti-phospho-IKKα/β, rabbit anti-IκBα, rabbit anti-p65, and rabbit anti-phospho-p65 antibodies (1:1000, Cell Signaling Technology, Danvers, MA, USA); and mouse anti-β-actin antibodies (1:8000, Sigma-Aldrich). The blots were then incubated with their respective secondary antibodies: horseradish peroxidase-conjugated goat anti-rabbit IgG, goat anti-mouse IgG (both 1:8000, Abcam), and donkey anti-goat IgG (1:5000, Santa Cruz Biotechnology). β-actin was used as the loading control. The immunoreactive bands were scanned using the Bio-Rad ChemiDoc™ XRS⁺ imager with Image Lab™ Software (Bio-Rad Laboratories, CA, USA). Band intensity was quantified using Quantity One software (Bio-Rad Laboratories).

Cheng P., Xie J., Liu Z, & Wang J. (2021). Aldose reductase deficiency inhibits LPS-induced M1 response in macrophages by activating autophagy. Cell & Bioscience, 11, 61.

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Chemidoc xrs imager

Lab products found in correlation

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279 protocols using chemidoc xrs imager

Methionine Inhibitor Fluorescent Labeling

Fluorescent PARP1 Protein Labeling

EPHX2 Inhibition and Labeling

Fluorescent PARP1 Protein Labeling

EPHX2 Inhibition and Labeling

Methionine Inhibitor Fluorescent Labeling

Western Blot Analysis of Inflammatory Signaling

Western Blot Analysis of Signaling Pathways

SDS-PAGE and Western Blotting Analysis

Western Blot Analysis of Protein Targets

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