All pre-treated samples were washed with PBS and lysed in lysis buffer (M-PER, Life Technologies, Waltham, MA, USA) plus protease inhibitor phenylmethylsulfonyl fluoride (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min on ice. The lysates were centrifuged at 12,000× g for 15 min at 4 °C. The supernatants were collected and the protein concentrations were determined using BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). After being heated for 5 min at 95 °C, equal proteins(30 µg) for each sample were separated by 10% SDS-polyacrylamide gel and then transferred to polyvinylidene difluoride (PVDF) membrane (Whatman, Lafayette, CO, USA) for 70 min at 100 V. The membrane was blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline-Tween 20 (0.1%) (TBS-T) for 1 h and incubated with anti-HDAC4 or anti-actin antibodies (Abcam, Cambridge, MA, USA) at 4 °C overnight. On the next day the membrane was incubated with anti-rabbit-Alexa Fluor 680 (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. The blots were scanned using an Odyssey fluorescence scanner (LI-COR Biosciences, Lincoln, NE, USA). The band intensity was quantified using the Odyssey software.
Alexa fluor anti rabbit 680
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor anti-Rabbit 680 is a fluorescent secondary antibody conjugate designed for use in immunoassays and other fluorescence-based applications. It is specific for rabbit primary antibodies and is labeled with the Alexa Fluor 680 fluorescent dye, which has an excitation maximum at 679 nm and an emission maximum at 702 nm.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey imager, by LI COR (5 mentions)
Odyssey fluorescence scanner, by LI COR (2 mentions)
Odyssey system, by LI COR (2 mentions)
Phospho akt ser473, by Cell Signaling Technology (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Anti mouse alexa fluor 680, by Thermo Fisher Scientific (2 mentions)
Sema3c, by Santa Cruz Biotechnology (2 mentions)
Tissuelyser 2, by Qiagen (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Anti mouse irdye 800cw, by LI COR (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
M per, by Thermo Fisher Scientific (1 mentions)
Whatman, by Cytiva (1 mentions)
Anti notch1, by Abcam (1 mentions)
Anti tubulin, by Santa Cruz Biotechnology (1 mentions)
Irdye 680lt goat anti mouse, by LI COR (1 mentions)
Donkey anti rabbit hrp, by Jackson ImmunoResearch (1 mentions)
Immobilon membrane, by Merck Group (1 mentions)
13 protocols using alexa fluor anti rabbit 680
1
Western Blot Analysis of HDAC4
2
Quantifying HDAC4 Expression in Chondrocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from human articular chondrocytes 48 h post-transfection of miR-365 mimic or inhibitor and their controls. Equal amounts of cell lysates were loaded in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane, as described previously [21 (link)]. Immunoblotting was coupled with fluorescent signal detection using an Odyssey fluorescence scanner (LI-COR Biosciences, Lincoln, NE, USA). The following antibodies were used for this study: anti-HDAC4 antibody and anti-tubulin antibody purchased from Santa Cruz biotechnology, anti-mouse-IRDye800 (Roche; Rockland Immunochemicals, Gilbertsville, PA, USA) and anti-rabbit-Alexa Fluor 680 (Molecular Probes, Eugene, OR, USA).
3
Chondrocyte Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein extracts of transfected chondrocytes or chondrocytes isolated from rib cage of miR‐146a−/− mice at age of P5 were prepared for Western blot analysis as previously described (Yang et al., 2016 ). The following antibodies were used for this study: anti‐NOTCH1 (Abcam); anti‐tubulin (Santa Cruz Biotechnology Inc., Santa Cruz, CA); and anti‐mouse‐IRDye800 and anti‐rabbit‐Alexa Fluor 680 (Molecular Probes, Eugene, OR).
4
Human PD-L1 Expression Quantification
5
Western Blot Sample Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell culture homogenates for western blot analyzes from were obtained by collecting the cells in ice-cold PBS 1×. Cells were homogenized with a 25G-syringe in lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2 mM sodium orthovanadate, 100 mM NaF, 20 mM sodium pyrophosphate, 1% NP-40, and protease inhibitors Mini protease tablet (Roche)) and centrifuging them at 700 × g for 10 min and 4 °C to remove nuclei, cell debris and floating cells. Mouse skeletal muscle, liver, kidney and heart tissues were homogenized in lysis buffer with a Minibeadbeater (Biospec) twice for 30 sec, incubated for 1 h at 4 °C in an orbital shaker, and then centrifuged for 15 min at 10,000 × g. Supernatants were aliquoted and kept at −20 °C.
Proteins from total homogenates were solved in 10%, 12.5%, or 15% acrylamide gels for SDS-PAGE and transferred to Immobilon membranes (Millipore). Primary antibodies used are specified in TableS1 . Secondary antibodies used included donkey anti-mouse HRP (715-035-150, Jackson Laboratories) and donkey anti-rabbit HRP (711-035-152, Jackson Laboratories), donkey anti-rat HRP (760–4457, Roche), goat anti-mouse DyLight 800 (SA535521, Invitrogen), goat anti-mouse IRDye 680LT (35518, Licor Biosciences), goat anti-rabbit DyLight 800 (SA535571, ThermoFisher), goat anti-rabbit Alexa Fluor 680 (A-21076, ThermoFisher).
Proteins from total homogenates were solved in 10%, 12.5%, or 15% acrylamide gels for SDS-PAGE and transferred to Immobilon membranes (Millipore). Primary antibodies used are specified in Table
6
Human PD-L1 Expression Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibody: Anti-human PD-L1 (E1L3N) (cell signaling technology, cat. 13684T). Secondary Antibody: Anti-Rabbit Alexa Fluor 680 (Thermo Fisher Scientific, cat. A10043).
7
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell extracts were prepared in 50 mM Tris-HCl, 150 mM NaCl, 1% NP40, 10 mM NaF, 10% Glycerol, supplemented with protease inhibitor cocktail (Roche, Cat. No. 04693116001) and quantitated using a BCA approach. 60 μg of protein, or 40 μl of conditioned media, was run on 10% acrylamide gels and transferred onto nitrocellulose membrane. Western blots were imaged on radiography film or by a LI-COR Odyssey system. actin or vinculin served as loading controls. Primary antibodies: phospho-Akt (Ser473; Cell Signaling Technology, Cat. No. 4060S), pan-Akt, (Life Technologies, Cat. No. 44609G), androgen receptor (Santa Cruz Biotechnology, Cat. No. sc-816), SEMA3C (Santa Cruz Biotechnology, Cat. No. sc-27796), GATA2 (Santa Cruz Biotechnology, Cat. No. sc-9008), FOXA1 (Santa Cruz Biotechnology, Cat. No. sc-6553), POU2F1 (Santa Cruz Biotechnology, Cat. No.s sc-232, sc-8024; Cell Signaling Technology, Cat. No. 4428S), actin (Sigma-Aldrich, Cat. No. A2066), and vinculin (Sigma-Aldrich, Cat. No. V4505). Secondary antibodies: anti-rabbit alexa fluor 680 (Invitrogen, Cat. No. A21109), anti-mouse alexa fluor 680 (Invitrogen, Cat. No. A21058), anti-goat alexa fluor 680 (Invitrogen, Cat. No. A21084), anti-rabbit HRP (Dako, Cat. No. P0448), anti-mouse HRP (Dako, Cat. No. P0447), and anti-goat HRP (Dako, Cat. No. P0160).
8
Western Blot Analysis of Prostate Cancer Markers
9
Immunoblotting Analysis of Metabolic Proteins
10
Quantifying Mitochondrial Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were homogenized in cell lysis buffer (30 mmol/L HEPES [pH 7.4], 1% NP-40, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 10% glycerol, and protease inhibitor cocktail [Sigma, St. Louis, MO]). The protein concentration was measured using a Micro BCA Protein Assay Reagent (Pierce, Rockford, IL). The protein extracts were resolved by SDS-PAGE and electrotransferred onto an Immobilon polyvinylidene fluoride membrane (Millipore, Bedford, MA). The membranes were probed with the following primary antibodies: MnSOD; Tom20 (Santa Cruz Biotechnology); succinate dehydrogenase subunit A (SDHA), phospho–dynamin-related protein (DRP1) (Ser637), DRP1, mitofusin 1 (MFN-1), TXNIP, GLUT1, GAPDH, and tubulin (Cell Signaling Technology, Danvers, MA); 4-hydroxynonenal (4-HNE) (Abcam, Cambridge, MA); and glutathione peroxidase 4 (GPX4) (R&D Systems). Alexa Fluor anti-rabbit 680 (Invitrogen, Carlsbad, CA) and anti-mouse 800 (VWR International, West Chester, PA) were used as the secondary antibodies. Fluorescence was quantified by using the Odyssey imager (LI-COR Biosciences, Lincoln, NE).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!