Proteins were extracted on ice using a RIPA lysis buffer (Beyotime, P0013C) containing protease inhibitors (Roche, 04693132001). Equal amounts of total proteins (60 µg) were mixed and dissolved in 4× SDS/PAGE sample buffer and heated to 100 °C for 5 min. The proteins were then separated on NuPAGE 10% Bis-Tris Gels (Invitrogen, NP0315BOX) and electrophoretically transferred onto polyvinylidene fluoride membranes. The membranes were immunoblotted overnight at 4 °C with the following antibodies: ARRB1 (Rabbit, Abcam, ab32099, 1:500), GAPDH (Mouse, Proteintech, 60004-1-Ig, 1:10,000). The following day, after washing three times with TBST, the membranes were incubated with conjugated fluorescent secondary antibody (Invitrogen), and the blots were imaged using a ChemiDoc™ Imaging Systems (Bio-Rad). The secondary antibodies used in the experiment included Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 800 (Invitrogen, A32730, 1:10,000) and Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 800 (Invitrogen, A32735, 1:10,000).
Alexa fluor plus 800
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor Plus 800 is a fluorescent dye developed by Thermo Fisher Scientific. It is designed for use in a variety of laboratory applications, including flow cytometry, immunoassays, and fluorescence microscopy. The dye has an excitation maximum at 784 nm and an emission maximum at 812 nm, making it suitable for detection in the near-infrared region of the spectrum.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor plus 680, by Thermo Fisher Scientific (5 mentions)
Alexa fluor plus 488, by Thermo Fisher Scientific (4 mentions)
Alexa fluor plus 555, by Thermo Fisher Scientific (4 mentions)
Image studio software, by LI COR (2 mentions)
Odyssey clx, by LI COR (2 mentions)
Alexa fluor 680, by Thermo Fisher Scientific (2 mentions)
Ibright imaging system, by Thermo Fisher Scientific (2 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (2 mentions)
α tubulin, by Merck Group (2 mentions)
Draq5, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (2 mentions)
Integrin β1, by Merck Group (2 mentions)
Antibodies to β actin and ha tag, by Cell Signaling Technology (2 mentions)
Antibodies against double stranded dna, by Abcam (2 mentions)
Histone h3 tri methyl k9, by Abcam (2 mentions)
Vamp3, by Proteintech (2 mentions)
Emerin, by Proteintech (2 mentions)
Apex1, by Proteintech (2 mentions)
Annexin a1, by Proteintech (2 mentions)
Mouse monoclonal antibody against human cd63 h5c6, by Developmental Studies Hybridoma Bank (2 mentions)
18 protocols using alexa fluor plus 800
1
Western Blot Analysis of Protein Expression
2
Quantitative Immunoblotting Analysis
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For immunoblotting, protein samples were separated by SDS–PAGE and transferred onto a nitrocellulose membrane using semidry blotting. Membranes were blocked in 4% milk in PBST (0.1% Tween‐20 in PBS) buffer for 30 min, followed by overnight incubation with the primary antibody diluted in 4% milk/PBST. After three washes in PBST, the membranes were incubated with secondary antibodies (goat anti‐mouse Alexa Fluor Plus 680 (Thermo Fisher Scientific, A32729) or goat anti‐rabbit Alexa Fluor Plus 800 (Thermo Fisher Scientific, A32735)) for 30 min at RT. Following three washes in PBST, the signal was detected by an Odyssey (LI‐COR) imaging system, quantified with the Image Studio software (LI‐COR), and plotted with the GraphPad Prism software.
3
Quantitative SARS-CoV-2 Protein Analysis
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After electrophoresis in NuPAGE 4 to 12% gels (Invitrogen), proteins were transferred to nitrocellulose membranes which were processed with the following antibodies: SARS-CoV-2 S1 (no. 40591-T62, Sino Biological), SARS-CoV-2 S2 (no. 40590-T62, Sino Biological), SARS-CoV-2 nucleoprotein (no. 35-579, ProSci), SARS-CoV-2 nsp1 (no. 10-500, ProSci), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; no. 66004-1-Ig, Proteintech), and β-tubulin (no. 66240-1-Ig, Proteintech). Secondary antibodies labeled with infrared dyes were obtained from Thermo Fisher Scientific: donkey anti-mouse, Alexa Fluor Plus 800 (no. A32789); donkey anti-mouse, Alexa Fluor Plus 680 (no. A32788); and goat anti-rabbit, Alexa Fluor Plus 680 (no. A32734). Membranes were scanned on Odyssey CLX (LI-COR Biosciences) and analyzed in Empiria Studio software (LI-COR).
4
Protein Complex Immunoprecipitation in S2 Cells
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S2 cells were transfected with pMT-N-EGFP, pMT-Flag-Ubi, and pMT-Dx-V5 (WT or ΔRF). After 48 h, 1 mM CuSO4 was added to induce protein expression, followed by incubation at 18°C for 24 h. The cells were lysed by lysis buffer (50 mM Tris-HCl, pH7.5, 125 mM NaCl, 5% glycerol, 0.5% NP-40, 1.5 mM MgCl2, 1 mM DTT, 1 mM EGTA, 1 mM N-ethyl-maleimide, 10 μM MG132, and Halt Protease and Phosphatase Inhibitor Cocktail [Thermo Fisher Scientific]), and pulled-down with GFP-Trap (ChromoTek). The immunoprecipitated samples and 10% lysates were separated on NuPage 3–8% Tris-Acetate gels (Thermo Fisher Scientific) and transferred to PVDF membranes (Merck). Primary antibodies used for Western blotting were rabbit anti-GFP (50430-2-AP, 1:5,000; Proteintech), mouse anti-Flag (M2, 1:5,000; Merck), mouse anti-V5 (OASA04489, 1:5,000; Aviva System Biology), and mouse anti-Peanut ((Cat# 4C9H4 anti-peanut, RRID:AB_528429, 1:10,000; DSHB), and staining was detected by LI-COR Odyssey imaging system with anti-mouse Alexa Fluor Plus 800 (Cat# A32730, RRID:AB_2633279; Thermo Fisher Scientific) and anti-rabbit Alexa Fluor Plus 680 (Cat# A32734, RRID:AB_2633283; Thermo Fisher Scientific) secondary antibodies, used at 1:10,000.
5
Immunofluorescence Profiling of Neuronal Targets
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The following primary antibodies were used in this study: mouse anti-EEA1 (1:25, BD Biosciences Cat# 610456), mouse anti-GABAB2 (1:400, Abcam ab181736), rabbit anti-GABAB2 (1:400, Abcam ab75838), rabbit anti-GABAB2N (1:250, custom-made by GeneScript, Benke et al., 2002 (link)), mouse anti-HA-tag (1:400, Sigma Aldrich, H9658), rabbit anti-NeuN (1:400, Millipore ABN78), rabbit anti-PP2A (1:400, MyBioSource MBS858915), mouse anti-Rab4 (1:25, BD Biosciences Cat# 610888) and rabbit anti-Rab7 (1:50, Abcam ab137029). Secondary antibodies: donkey anti-rabbit AlexaFluor Plus 488 (1:2000, Thermo Fisher Scientific A32790), donkey anti-mouse AlexaFluor Plus 555 (1:2000, Thermo Fisher Scientific A32773), goat anti-rabbit AlexaFluor Plus 800 (1:2000, Thermo Fisher Scientific A32735), and AlexaFluor 488-conjugated streptavidin (1:500, Jackson ImmunoResearch 016-540-084). All normal laboratory chemicals used were purchased from Sigma-Aldrich.
6
GFP and Hywi/Hyli Protein Detection
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To detect GFP protein in cnnos1::GFP animals, 10 Hydra were homogenized in SDS-PAGE sample buffer for immunoblot analysis. Anti-GFP antibody was used at a 1:1000 dilution (Sigma, catalog #11814460001, Roche). All antibodies were diluted in blocking solution (3% w/v powdered milk dissolved in 0.1% TBS-Tween). For detection of Hywi and Hyli protein, 8 µg of purified anti-Hywi or anti-Hyli antibody in 50 mL of blocking solution was used for immunoblots. Anti-GFP was detected using goat anti-mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 800 (ThermoFisher Scientific, A32730). Anti-Hywi was detected using goat anti-Rabbit IgG (H + L) Superclonal Secondary Antibody, Alexa Fluor 680 (Thermofisher Scientific, A27042). Anti-Hyli was detected using goat anti-guinea pig IgG (H + L) Cross Adsorbed Secondary Antibody, DyLight 800 conjugate (ThermoFisher Scientific, SA5-10100). All secondary antibodies were diluted 1:10,000 in blocking solution. Immunoblots were imaged using the LI-COR Odyssey platform.
7
Quantitative Analysis of Filamin A Expression
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Cells were cultured in a 6 well plate for 72h after stimulation with doxycycline. Lysis, gel and blotting were performed as described previously[13 (link)]. For the detection, the primary antibodies for filamin A (Thermo Fisher, MA5-11705) and tubulin (Sigma, T 9026) were used and diluted by manufacturer’s instructions. For secondary antibody detection, ALEXA FLUOR PLUS 800 (Thermo Fisher, A32730) were used, imaged with the Odyssey Fa and analyzed and quantified by Image Studio Software (LiCor).
8
Western Blot Analysis of Prolyl Hydroxylase Enzymes
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Islets were isolated from mice, and then 10 µg of protein was loaded on a gel. After protein transfer to a PVDF membrane, proteins were labeled using a no-stain protein labeling kit (ThermoFisher Scientific, A44449) (see Fig. 1 (20 ) for a representative total protein normalization blot). The PVDF membrane was then assessed for PHD1, PHD2, or PHD3 expression. Primary antibodies: rabbit anti-PHD1 (RRID:AB_10861175, Abcam), rabbit anti-PHD2 (RRID:AB_568562, ThermoFisher Scientific), rabbit anti-PHD3 (RRID:AB_2293343, ThermoFisher Scientific). They were then incubated with goat antirabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 800 (RRID:AB_2633284, ThermoFisher Scientific, A32735). Blots were imaged using an iBright imaging system (ThermoFisher Scientific), and then band intensities were normalized to total protein levels using the iBright™ Analysis Software (ThermoFisher Scientific).
9
Immunoblotting with Cell Lysates
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Tissue lysates used for immunoblotting were prepared in Cell Lysis Buffer (Cell Signaling Technologies) containing protease inhibitor tablets (Roche), phosphatase inhibitor cocktails 2 and 3 (Sigma), and 10 mM PMSF. 50 μg of protein was loaded onto a 4–12% Bis-Tris gel (Novex), subjected to SDS-PAGE, and then transferred onto PVDF membranes. Membranes were blocked and then probed with the appropriate antibodies. All primary antibodies were used at a concentration of 1:1000. Secondary antibodies were diluted 1:10000. The following antibodies were used: S6 (Cell Signaling Technologies #2317), phospho-S6 (Ser 235/236) (Cell Signaling Technologies #4858), 4E-BP1 (Cell Signaling Technologies #9644), phospho-4E-BP1 (Thr 37/46) (Cell Signaling Technologies #2855), pan-actin (Cell Signaling Technologies #8456), puromycin (EMD Millipore #MABE343), β-tubulin (Sigma Aldrich, T8328), Alexa-Fluor Plus 800 (Thermo Fisher, A32735), and Alexa-Fluor Plus 594 (Thermo Fisher, A32742). Immunoblots were developed using a Li-Cor Odyssey CLx and quantified using the Li-Cor software.
10
Antibody Characterization for Neuron Analysis
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The following primary antibodies were used in this study: Rabbit anti NeuN (1:400 for microscopy, 1:1000 for Odyssey Scanner, Millipore Cat# ABN78), mouse anti GABAB2 (1:400 for microscopy, abcam Cat# ab181736), rabbit anti GABAB2 (1:2000 for Odyssey Scanner, abcam Cat# ab75838), mouse anti EEA1 (1:500, BD Biosciences Cat# 610456), mouse anti Rab11 (1:100, Millipore Cat# 05-853), and goat anti Rab7 (1:50, Santa Cruz Cat# sc-11303). Secondary antibodies: donkey anti rabbit Alexa Fluor Plus 488 (1:2000, Thermo Fisher Scientific Cat# A32790), donkey anti mouse Alexa Fluor Plus 555 (1:2000, Thermo Fisher Scientific Cat# A32773), and goat anti rabbit Alexa Fluor Plus 800 (1:2000, Thermo Fisher Scientific Cat# A32735). All chemicals used for electrophysiology solutions were purchased from Sigma-Aldrich and baclofen was purchased from Tocris Bioscience.
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