This assay was carried out based on the method of Bolt and Mahoney [23 (link)]. Total head homogenates of 25 flies of each group underwent a standard protein extraction process. Following SDS-PAGE, the gel was transferred to the blotting unit, and after protein bands transfer onto the nitrocellulose membrane, the blot was blocked with 3% BSA at 4°C overnight. Then the blot was kept for incubation with 1:5000 anti-human α-synuclein mouse IgG monoclonal antibody (Cat # ABH0261, Invitrogen, USA), at 4°C overnight. The blot was washed with 0.3% phosphate buffer saline Triton X (PBSTx) for the duration of 30 min. Next, the blot was incubated with 1:1,000 diluted buffer of secondary rabbit anti-mouse HRP conjugated antibody (Cat # 6728, Abcam, UK) in 1X PBS for 1 h at room temperature. The blot was given a wash and then developed using a chromogenic substrate (1X TMB) to detect the horseradish peroxidase (HRP). The expression of the test protein can be monitored by the development of bluish-green color bands to which secondary antibody is bound. Anti-ß-actin antibody (Cat # 8224, Abcam, UK) was utilized as a loading control.
Anti actin antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-actin antibody is a laboratory reagent used to detect and quantify the presence of actin, a protein found in eukaryotic cells. It is a widely used tool in cell biology research and can be employed in various techniques, such as western blotting, immunocytochemistry, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Merck Group (2 mentions)
Alexa fluor antibodies, by Jackson ImmunoResearch (2 mentions)
Ir dye labeled reagents, by LI COR (2 mentions)
Fetal bovine serum (fbs), by Atlas Biologicals (2 mentions)
Anti creb antibody, by Merck Group (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Anti flag antibody, by Abcam (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti rankl, by Abcam (2 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Anti ldha antibody, by Cell Signaling Technology (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Anti jak1, by Cell Signaling Technology (1 mentions)
Irdye 680rd donkey anti mouse igg secondary antibody, by LI COR (1 mentions)
Irdye 800cw donkey anti rabbit igg secondary antibody, by LI COR (1 mentions)
47 protocols using anti actin antibody
1
Quantitative Western Blot Analysis of α-Synuclein
2
Glycolytic Enzyme Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lines underwent protein extraction using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA). Protein concentrations were determined by Pierce BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA). The proteins in equivalent amounts (10–40 μg/well) were separated by electrophoresis in a NuPAGE gradient 4–12% Bis-Tris Gel (Invitrogen, Carlsbad, CA, USA) and were immuno-blotted with anti-LDH-A antibody (Cell Signaling Technology, Danvers, MA, USA) at a 1:1,000 dilution and anti-ß-actin antibody (Abcam Inc., Cambridge, MA, USA) at a 1:5,000 dilution antibodies. In addition, we used Hexokinase I and II (Cell Signaling Technology Inc., Danvers, MA, USA) to detect the expression of these glycolytic enzymes as described by the protocol. Immune complexes were detected by horseradish-peroxidase-labeled antibodies and enhanced chemiluminescence reagent (Amersham, Buckinghamshire, UK).
3
Comprehensive Genomic and Proteomic Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence staining: anti-γH2A.X (Cell Signaling Technology, 2577), anti-Rad51 (Abcam, ab133534) and goat anti-Rabbit IgG secondary antibody, Alexa Fluor 594 (Invitrogen, A11037). Western blot: anti-MBD3 antibody (Abcam, ab91458), anti-Histone H3 antibody (Abcam, ab24834), anti-ß Actin antibody (Abcam, ab8227), IRDye® 800CW Donkey anti-Rabbit IgG secondary antibody (Licor, 926-32213) and IRDye® 680RD Donkey anti-mouse IgG secondary antibody (Licor, 926-68072). ChIP: anti-Histone H3 (acetyl K27) antibody (Abcam, ab4729), anti-Histone H3 (acetyl K9) antibody (Abcam, ab12179), normal Rabbit IgG Control (R&D, AB-105-C) or anti-mouse IgG (Molecular Probes, 6691-1). Immunohistochemistry: anti-Histone H3 (acetyl K27) antibody (Abcam, ab177178), anti-Histone H3 (acetyl K9) antibody (Abcam, ab32129), anti-FGF10 antibody (Genetex, GTX12469) and anti-JAK1 (Cell Signaling Technology, 3344).
4
Western Blot Analysis of LRP1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aag2 cells were lysed using RIPA buffer containing a protease inhibitor cocktail (Sigma). Protein concentration was determined using a BCA assay. 9 µg of total protein from each sample was separated by a SDS-PAGE gel (5–9%) and transferred to a nitrocellulose membrane. Membranes were blocked with 5% non-fat dry milk at room temperature for 1 hour prior to overnight incubation at 4 °C with primary antibody anti-LRP1 (cat. no. ab 92544, 1:20000, Abcam) and anti-actin antibody (cat. No. SC-1615, 1:1000, Santa Cruz Biotechnology) as a loading control. The membranes were washed with PBST and incubated at room temperature for 1 hour with anti-rabbit IgG-HRP (cat. no. 70745, 1:2000, Cell Signaling Technology) and anti-goat IgG-HRP linked (cat. no. ab6741, 1:2000, Abcam) secondary antibodies. Signal was detected using a ChemiDoc Touch Imaging System by Biorad.
5
Transgenic Plant Protein Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The anther tissues of transgenic plants were ground in RIPA buffer and placed on ice for 35 min for protein extraction. The extracted proteins were added to the protein loading buffer, heated at 95°C, and separated on a 15% SDS-PAGE gel for WB. Agrisera rabbit anti-Atg8 antibody and Abcam anti-actin antibody were used in this assay.
6
Resveratrol Autophagy Modulation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Resveratrol, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), 3-Methyladenine (3-MA), Chloroquine (CQ), 4′,6-Diamidino-2-phenylindole (DAPI), poly-L-lysine, Benzo[a]pyrene (BaP), Cadmium chloride, and 3,3′,5,5′ Tetrametyl-benzidine (TMB) were purchased from Sigma-Aldrich (St. Loius, MI, USA). The anti-LC3 antibody was from Sigma-Aldrich and the antibodies against AMPK, phospho-AMPK (Thr 172), acetyl-CoA carboxylase (ACC), and phospho-ACC (ser 79) were from Cell Signaling Technology (Danvers, MA, USA). Anti-COX-2 antibody, anti-p62 antibody, and anti-actin antibody were purchased from Abcam (Cambridge, MA, USA), Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), and Young In Frontier Co., Ltd. (Seoul, South Korea), respectively.
7
Verifying FoAPY1 Protein Secretion in Fusarium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to verify the secretion function of the FoAPY1 protein in F. oxysporum, the strains of FoAPY1-Flag and FoAPY1∆sp-Flag were cultured in PDB liquid medium for obtaining the conidia. Then, the conidia of two strains and tomato roots were co-cultured, respectively, in 10% YEPD liquid medium at 25°C and 180 rpm for 16 h. The total proteins in culture supernatants were collected after centrifugation using precipitating methods by adding 20% acetone (w/v) and then stored at-80°C for 12 h. Next, the mixed solution was centrifuged at 12,000 g at 4°C for 30 min. The total proteins were dissolved in 1× protein loading buffer and then boiling for 10 min. The target proteins were detected by western blotting using anti-flag antibody (1: 10,000, abcam). The anti-actin antibody (1: 5,000, abcam) was used for checking the possibility of the cell lysis during the mycelia growth.
8
Western Blot Analysis of PDGFR-β
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates (30-60 μg/lane), as determined by BCA protein assay (Life Technologies), were separated in 4% to 12% SDS-PAGE (Life Technologies) and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were blocked with 5% nonfat dried milk in TBS (KPL, Gaithersburg, MD)-Tween 20 (Sigma Aldrich) (20 mm Tris-HCI, pH 7.5; 8 g/l of sodium chloride; 0.1% Tween 20). Blots were incubated with antibodies against total PDGFR-β and phospho-PDGFR-β (tyr751) (Cell Signaling Technology, Beverly, MA) at a 1:1000 dilution. Antiactin antibody from Abcam Inc. (Cambridge, MA) was used as a loading control. Bands were visualized on camera using West Femto ECL detection reagent (Life Technologies).
9
Quantifying PAX3 in Melanocytes and Melanoma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neonatal and adult melanocyte cells and primary and metastatic melanoma cells were lysed using cell lysis buffer (50 mM pipes, 85 mM KCl, 1% Nonidet P-40, protease inhibitor cocktail (PIC, Roche), pH 8.0). Following centrifugation the supernatant (containing cytosolic proteins) was removed and nuclear proteins were extracted with nuclear lysis buffer (50 mM Tris, 10 mM EDTA, 1% SDS, 1× PIC, pH 8.0). 20 μg Total nuclear protein (20 μg) was loaded onto NuPAGE 4–12% Bis-Tris gels (Life Technologies) and separated by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes (Bio-Rad), probed with mouse monoclonal PAX3 antibody (DSHB, 1/1000) and visualised using the Westernbreeze chemiluminescent detection kit (Life Technologies) as per the manufacturer’s instructions. To quantify PAX3 protein levels, membranes were stripped using Restore Western Blot Stripping Buffer (Thermo Scientific) and re-probed with anti-actin antibody (rabbit polyclonal, Abcam, 1/1000). The levels of each protein were assessed by densitometry using the GS-800 Calibrated Densitometer (Bio-Rad). PAX3 protein levels were compared between melanocyte cells and primary or metastatic melanoma cell lines using a one-way ANOVA.
10
TRIM14 Protein Purification Protocol
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!