Western blot analyses were performed on the same protein samples as those used for the AChE assay. Twenty micrograms of each protein were separated by 12% sodium-dodecyl-sulfate-polyacrylamide gels and electrotransferred onto polyvinylidene fluoride membranes using a Bio-Rad miniature transfer apparatus for 100 min at 0.3 A. The membranes were then blocked using a blocking buffer (5% non-fat dry milk in PBS containing 0.05% Tween 20) for 1 h at room temperature (25 °C). The membranes were then incubated with primary antibodies overnight at 4 °C with rabbit monoclonal anti-brain-derived neurotrophic factor (BDNF, 1:1000; Abcam, Cambridge, UK) and mouse monoclonal anti-ß-actin (1:10000; Sigma-Aldrich). The corresponding secondary antibodies were then applied for 90 min at room temperature with goat anti-rabbit IgG(H+L)-HRP (1:2000 at BDNF; GenDEPOT, Katy, TX, USA) and goat anti-mouse IgG(H+L)-HRP (1:10000; GenDEPOT). The proteins were visualized using an enhanced chemiluminescence solution (WEST-Queen western blot detection kit, iNtRON Biotechnology), and blots were analyzed using a LAS 4000 mini (GE Healthcare Life Sciences). The intensity of each band was measured using optical densitometry of the analysis system (Multi Gauge version 3.0; Fujifilm, Tokyo, Japan).
Mouse monoclonal anti actin
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
Mouse monoclonal anti-actin is a laboratory reagent used to detect and quantify the presence of actin, a ubiquitous cytoskeletal protein, in biological samples. It provides a specific and reliable method for identifying and characterizing actin in various cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 goat anti mouse igg antibody, by Thermo Fisher Scientific (2 mentions)
Hybond ecl nitrocellulose membrane, by GE Healthcare (2 mentions)
Phosphatase inhibitor, by Merck Group (2 mentions)
Protease inhibitor mixture, by Merck Group (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Gel electrophoresis reagents, by Bio-Rad (2 mentions)
Alexa fluor plus 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor plus 488 conjugated anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal anti β catenin, by BD (2 mentions)
Mouse monoclonal anti gfp, by Roche (2 mentions)
Mouse monoclonal anti cytochrome c, by Santa Cruz Biotechnology (2 mentions)
Rabbit polyclonal anti bkβ1, by Abcam (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Miniature transfer apparatus, by Bio-Rad (1 mentions)
Goat anti rabbit igg h l hrp, by GenDEPOT (1 mentions)
Goat anti mouse igg h l hrp, by GenDEPOT (1 mentions)
48 protocols using mouse monoclonal anti actin
1
Western Blot Analysis of BDNF Protein
2
Western Blot Validation of 2-DE Data
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The validation of the 2-DE data was carried out using Western blot analysis. To assure for the reproducibility of the Western blot analysis, at least three biological and experimental replicates were performed. 40 μg proteins were separated by SDS-PAGE and transferred to Hybond ECL nitrocellulose membrane (GE Healthcare). Immunodetection was performed according to Towbin et al. [24 (link)]. Briefly, membranes were blocked in 5% milk for 2 h at room temperature, followed by overnight incubation at 4°C with diluted specific primary antibody. Mouse monoclonal anti-CRABP1 (1:1000) (abcam), rabbit anti-Cofilin (1:1000) (sigma), mouse monoclonal anti-StAR (1:250) (abcam) and mouse monoclonal anti-ß-actin (1:5000) (sigma) were used as primary antibodies. Molecular Probes Alexa Fluor 647 goat anti-mouse IgG antibody or Alexa Fluor 647 goat anti-rabbit IgG (1:2000) were used as secondary antibodies. Before imaging, the blots were dried in the dark. The blot membranes were scanned at 50 μm resolution on a Fuji FLA-5100 scanner (Fuji Photo) with single laser-emitting excitation light at 635 nm.
3
Western Blot Analysis of Chromatin Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins (40 μg) were separated by SDS-PAGE and transferred to Hybond ECL nitrocellulose membrane (GE Healthcare). Immunodetection was performed according to Towbin et al.71 (link). Mouse monoclonal anti-Trim28/Kap1 [20C1] antibody ChIP Grade (Abcam), rabbit monoclonal anti- Cbx5/HP1 alpha antibody (Abcam)/ goat polyclonal to Cbx5/HP1 alpha-ChIP Grade (Abcam), rabbit polyclonal to Cbx1/HP1 beta (Abcam), rabbit polyclonal to Cbx3/HP1 gamma (Abcam), mouse monoclonal anti-ß-actin (Sigma) (1:5000), rabbit polyclonal anti-H3K9me3 ChIP grade (Abcam) and Gapdh (HyTest, Turku, Finland) were used as primary antibodies. Molecular Probes Alexa Fluor 647 goat anti-mouse IgG antibody or Alexa Fluor 647 goat anti-rabbit IgG (1:2000) were used as secondary antibodies. Before imaging, the blots were dried in the dark. The blot membranes were scanned at 50 μm resolution on a Fuji FLA-5100 scanner (Fuji Photo) with single laser-emitting excitation light at 635 nm.
4
Western Blot Analysis of Cyclin D1 and Rb
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in lysis buffer containing 62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, and 50 mM diethylenetriaminepentaacetic acid (DTT). After sonication, protein concentration was determined and 50 µg protein was subjected to SDS-PAGE, transferred to nitrocellulose membranes, blocked with 5% milk, and incubated with primary antibodies (1:1000 anti-cyclin D1: #2926 Cell Signaling; 1:500 mouse monoclonal anti-retinoblastoma protein: BD Biosciences; 1:10,000 mouse monoclonal anti-ß-actin: Sigma) and HRP-conjugated secondary antibodies. Bands were visualized using SuperSignal West Pico Chemiluminescent Substrate from Pierce.
5
Immunoblot Analysis of Antiviral Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbit anti-ADAR1 D7E2M antibody (1:1000, Cell Signaling), mouse monoclonal anti-MAVS E3 (1:250, Santa Cruz), mouse monoclonal anti-RNase L (1:1000) (Dong and Silverman, 1995 (link)), mouse monoclonal anti-PKR B-10 antibody (1:200, Santa Cruz), mouse monoclonal anti-GAPDH GA1R (1:1000, Thermo-Fisher), mouse monoclonal anti-NS2 (1:600) (provided by Stuart Siddell, University of Bristol, United Kingdom (Gusho et al., 2014 (link)) were used to detect ADAR1, MAVS, RNase L, PKR, GAPDH and NS2 respectively. Mouse monoclonal anti-Flag M2-antibody (1:5000) and mouse monoclonal anti-ß-actin (1:50000) were from Sigma-Aldrich. Rabbit anti-eIF2α (1:1000) or anti-Ser-51-phosphorylated eIF2α (1:1000) were from Cell Signaling Technology. Secondary antibodies were goat anti-mouse antibody (1:5000, Santa Cruz) and goat anti-rabbit antibody (1:3000, Cell Signaling) conjugated with HRP(Horseradish peroxidase).
6
Immunoblot Analysis of FcgR Receptors
7
Antibodies for Insulin Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-IRS1 rabbit polyclonal antibody against IRS1 (1:500, #06-248) was purchased from Merck Millipore (Billerica, MA, USA); IRS2 rabbit polyclonal antibody (1:1000, #4502) was obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); IR rabbit monoclonal antibody (1:5000, ab5500) was obtained from Abcam (Cambridge, UK); IGF1 rabbit monoclonal antibody (1:1000, #3018) was obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); tubulin mouse monoclonal antibody, #sc-8035) was obtained from Santa Cruz Biotech (CA, USA); and mouse monoclonal anti-ß-actin (1:2000, #A2228) was from Sigma Aldrich (MO, USA).
Secondary antibodies linked to horseradish peroxidize (anti-mouse #122011 and anti-rabbit #122825) were purchased from Jackson Immuno Research Laboratories, Inc. (Baltimore, PA).
Secondary antibodies linked to horseradish peroxidize (anti-mouse #122011 and anti-rabbit #122825) were purchased from Jackson Immuno Research Laboratories, Inc. (Baltimore, PA).
8
Phospho-rpS6 Signaling in Activated T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SDS-PAGE was performed on protein extracts obtained from human CD4+ T cell activated in vitro as previously described. Western blotting was performed as previously described (Ricciardi et al., 2018 (link)). The following antibodies were used for Western blotting: rabbit polyclonal to phospho-rpS6 Ser235/236 (1:1000, Cell Signaling), rabbit polyclonal to phospho-rpS6 Ser240/244 (1:1000, Cell Signaling), mouse monoclonal anti-Vinculin (1:1000, Millipore), mouse monoclonal anti-Actin (1:1000, Sigma), mouse monoclonal anti-Puromycin (1:10000, Millipore). Chemiluminescent signals were detected using Amersham ECL Prime (GE Healthcare Life Sciences) and images were acquired using the iBright CL750 Imaging System (Thermo Fisher Scientific).
Where indicated, cells were treated with either 2 μM PP242 (Sigma-Aldrich) or 3 μM MNK inhibitor (Sigma-Aldrich) for 30 min after 48 hr of Dynabeads stimulation.
Where indicated, cells were treated with either 2 μM PP242 (Sigma-Aldrich) or 3 μM MNK inhibitor (Sigma-Aldrich) for 30 min after 48 hr of Dynabeads stimulation.
9
Western Blot Quantification Procedure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured cells were collected in PBS-EDTA (5 mM) before centrifugation at 3,000 rpm for 5 min. Pellets were lysed in RIPA buffer with a complete proteases inhibitor, then centrifuged at 13,000 rpm for 10 min. Total protein concentration was assessed in the supernatant by Bradford method. Each sample (50 μg) was resuspended in Laemli buffer (1x final) and boiled at 96 °C for 5 min before loading and resolving on 10% SDS-PAGE gel. Wet transfer was done using Hybond-C membrane (Amersham Bioscience) at 100 V for 1 h30, then membrane were blocked with 5% PBS-milk solution for 1 h15 and blotted overnight with following antibodies: Mouse monoclonal 3D5 anti-Bace1 (kindly given by Dr. Vassar R.), rabbit polyclonal anti-XBP-1 (M-186 Santa-Cruz Biotechnology), mouse-monoclonal anti-actin (Sigma Aldrich), mouse-monoclonal anti-Myc (9E10; Santa-Cruz biotechnology). Protein immunoreactivity was assayed using peroxidase-coupled antibodies (Jackson immunoresearch), resulting electrochimio-luminescence was detected with luminescence analyzer LAS-3000 (Raytech). Multi-Gauge software (FUJI film) was used for image protein quantification. All densitometric quantifications were normalized using actin as loading control.
10
Generation of Rabbit Anti-COX-1 Antibody
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Generation of a new rabbit polyclonal anti-COX-1 antibody was conducted at the Vanderbilt Antibody and Protein Resource. The protein sequence of human COX-1 and COX-2 were compared to identify unique COX-1 sequences [22 ]. Anti–human COX-1 antibodies were generated by immunization of two New Zealand white rabbits with three KLH-conjugated human COX-1 specific peptides (peptide sequences: CQDDGPAVERPS, ADPGAPTPVC, and LMHYPRGIPPQSQMAC) which have no overlap with the corresponding COX-2 protein sequence (Additional file 1 ). Bleeds from sequential boosts were collected from both rabbits and tested by Western blot and ELISA. Antisera from both animals were mixed for affinity purification. Polyclonal antibodies were purified using affinity against immunizing peptides and then the elution was sequentially cleared by a mouse COX-2 immobilized affinity column. Final antibodies were further validated by Western blot, and demonstrated to have no cross-reactivity with COX-2 (Additional file 1 ). Antibodies used for Western blot included the newly generated Vanderbilt rabbit polyclonal anti-COX-1 (1:2000 overnight), rabbit polyclonal anti-COX-2 (1:1000 overnight; Cayman Chemicals, Ann Arbor, MI), and mouse monoclonal anti-actin (1:10000 for 15 mins; Sigma Chemical Co, St Louis, MO); positive controls included recombinant ovine COX-1 and human COX-2 (both from Cayman Chemicals).
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!