Brain tissues were first homogenized in buffer H, followed by protein concentration determination (Bio-rad). Samples were then separated by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with antibodies against ADCY1 (Sigma, 1:1000), pERK½ (Cell Signaling, 1:2000), ERK½ (Cell Signaling, 1:1000), and β-actin (Sigma, 1:10000) overnight at 4 °C. Immuno signal was detected with IRDye 800CW-conjugated goat anti-rabbit or IRDye 680RD-conjugated goat anti-mouse antibody (LI-COR, 1:5000) using the Odyssey Imaging system. The signal intensity of the detected protein was quantified using ImageJ (NIH, MD, USA).
Irdye 800cw conjugated goat anti rabbit
Manufactured by LI COR
Sourced in United Kingdom
IRDye 800CW-conjugated goat anti-rabbit is a secondary antibody conjugated with the near-infrared dye IRDye 800CW. It is designed for use in various fluorescence-based detection and imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (3 mentions)
Blocking buffer, by Rockland Immunochemicals (2 mentions)
Irdye 680lt conjugated goat anti mouse, by LI COR (2 mentions)
Mouse anti tubulin t5168, by Merck Group (2 mentions)
Odyssey imaging system, by LI COR (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
Protein concentration determination, by Bio-Rad (1 mentions)
β actin, by Merck Group (1 mentions)
Irdye 680rd conjugated goat anti mouse antibody, by LI COR (1 mentions)
Immobilon transfer membrane, by Merck Group (1 mentions)
Rabbit anti phosphorylated akt, by Cell Signaling Technology (1 mentions)
Ab175349, by Abcam (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
α gapdh, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Irdye 680rd conjugated goat anti mouse, by LI COR (1 mentions)
7 protocols using irdye 800cw conjugated goat anti rabbit
1
Western Blot Analysis of ADCY1, pERK1/2, and β-Actin
2
Immunoblot Analysis of Phosphorylated Akt and p38
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After 120 min stimulation of B cells isolated from healthy donors by anti-IgG/IgM antibody with or without pre-IFN treatment, cells were lysed with 1x SDS sample buffer (Cell Signaling Technologies) and sonicated. The cell lysates were electrophoresed on 12% polyacrylamide gel and then transferred onto Immobilon transfer membranes (Millipore). The membranes were treated with rabbit anti-phosphorylated Akt (Cell Signaling Technologies), rabbit anti-phoshorylated p38 (Cell Signaling Technologies), and rabbit anti-tubulin antibody (abcam, Cambridge, UK). IRDye 800CW-conjugated goat anti-rabbit (LI-COR Biosciences, Lincoln, NE) was used as a secondary antibody. Fluorescence intensity was measured, and densitometric analysis was performed with an Odyssey Infrared Imaging System (LI-COR Biosciences).
3
Western Blotting of Adult Brain
4
Quantifying Synaptic Protein Levels
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Western blotting of adult head lysates (three heads per sample with one head loaded per lane) was performed using standard laboratory procedures with mouse anti-Tubulin (T5168; Sigma) at 1:10000 (UAS rescue experiments) or 1:1000000 (CRISPR experiments) and rabbit anti-Cpx at 1:5000. IRDye 680LT-conjugated goat anti-mouse, 1:5000 (926–68020; LICOR) and IRDye 800CW conjugated goat anti-rabbit, 1:5000 (926–32211; LICOR) were used as secondary antibodies. Blocking was performed in a solution containing four parts TBS (10 mM Tris Base pH 7.5, 150 mM NaCl) to one part Blocking Buffer (Rockland) for one hour. Antibody incubations were performed in a solution containing four parts TBST (1X TBS with 1% Tween-20) to one part Blocking Buffer. A LI-COR Odyssey Imaging System (LI-COR Biosciences, Lincoln, MA, USA) was used for visualization and analysis was performed using FIJI image analysis software. Relative Cpx expression was calculated by normalizing to Tubulin intensity.
5
Western Blot Analysis of GATA6 and ACE2
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Cells were lysed in RIPA Lysis Buffer (Merck 20-188) in the presence of cOmplete protease inhibitor cocktail (Roche 11697498001). Lysates were nutated at 4 °C for 10 min, then centrifuged at 20,000 × g for 15 min at 4 °C. Equal amounts of cell lysates were denaturated in 4× Laemmli sample buffer (Bio-RAD #1610747), separated on 4–12% NuPAGE Bis-Tris gels (invitrogen), blotted onto nitrocellulose membranes and immunoblotted with primary antibodies αGATA6 (Abcam ab175349, 1:500, ab22600, 1:500), αGAPDH (Cell Signaling 14C10, 1:2000), αACE2 (Sino Biological #10108-T60, 1:1000). The secondary antibody used was IRDye® 800CW conjugated Goat anti-Rabbit (Licor, 1:20,000). Reactive bands were detected by Odyssey CLx infrared imaging system (Licor). Protein concentration was measured by the BCA protein assay kit (Pierce 23225). Protein quantification was performed on Licor software. Uncropped and unprocessed scans are presented in the source data file.
6
Cytokine Signaling in MDA-MB-468 Cells
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MDA-MB-468 cells were seeded and serum-starved for 24 hours. They were subsequently treated with TNF-α (20 ng/ml), IL-6 (20 ng/ml), EGF (10 ng/ml), IFN-α (20 ng/ml), or IFN-γ (40 ng/ml) for 30 min. Whole-cell lysates were generated by lysis with radioimmunoprecipitation assay buffer, along with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific). For cell fraction, the cells were lysed using the Pierce subcellular fractionation kit (PI78840). SDS-PAGE gels were run at 120 V for 75 min, transferred onto polyvinylidene difluoride membranes (#IPFL00010, Millipore, Billerica, MA) at 70 V for 90 min. Membranes were blocked with LI-COR blocking buffer (#927-40000) for 1 hour at RT and then subsequently probed with primary antibodies diluted at 1:1000 in 5% BSA/tris-buffered saline–Tween 0.1% (TBST) overnight at 4°C. Incubation with secondary antibodies for 1 hour at RT containing fluorophores at 1:20,000 dilution (IRDye 800CW conjugated goat anti-rabbit; #926-32211, LI-COR Biosciences, Lincoln, NE) enabled visualization on the Odyssey Infrared Imaging System from LI-COR Biosciences. Washes in between incubations were done for 10 min × 3 using TBST.
7
Western Blot Protein Detection Protocol
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