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Rabbit anti actin antibody

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Rabbit anti-actin antibody is a laboratory reagent used to detect and quantify the presence of actin, a ubiquitous and essential structural protein found in all eukaryotic cells. This antibody is raised in rabbits and specifically binds to actin, allowing for its identification and measurement in various experimental applications.

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Lab products found in correlation

Pvdf membrane, by Bio-Rad (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions) Protease inhibitor mixture, by Merck Group (2 mentions) Mab810, by Merck Group (2 mentions) Ibright fl1500 imaging system, by Thermo Fisher Scientific (2 mentions) Amersham protran 0.45 μm nitrocellulose membrane, by GE Healthcare (2 mentions) Affinipure f ab 2 fragment goat anti mouse igg, by Jackson ImmunoResearch (2 mentions) Goat anti rabbit igg whole molecule peroxidase antibody, by Merck Group (2 mentions) Cdp star chemiluminescence reagent, by PerkinElmer (2 mentions) Goat anti il 32 af3040 antibody, by R&D Systems (2 mentions) Clarity western ecl blotting substrate, by Bio-Rad (2 mentions) Nitrocellulose membrane, by Bio-Rad (2 mentions) Protein assay kit, by Bio-Rad (2 mentions) M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Polyacrylamide gel, by Bio-Rad (2 mentions) Horseradish peroxidase conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions) G box chemi xx6 gel documentation system, by Syngene (1 mentions) Enhanced chemiluminescence substrate, by Bio-Rad (1 mentions)

59 protocols using rabbit anti actin antibody

1

Immunoblotting for TBX3 Protein

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Cell pellets were lysed in RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl; 1% (vol/vol) IGEPAL CA-630, 0.5% (vol/vol) sodium deoxycholate, 0.1% (wt/vol) SDS, 1 mM DTT, protease inhibitor cocktail) and clarified by centrifugation to remove cell debris. Forty micrograms of lysate supernatants was separated by SDS-polyacrylamide gel electrophoresis, electroblotted onto PVDF membranes by semi-dry transfer (Bio-Rad), and blocked in blocking buffer (1% (wt/vol) casein, 0.1% (vol/vol) Tween 20, PBS). TBX3 was detected with 1 μg/ml rabbit anti-TBX3 antibody (Thermo Fisher Scientific) and actin with 400 ng/ml of rabbit anti-actin antibody (Sigma-Aldrich). Primary antibodies were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling). Detected proteins were visualized with enhanced chemiluminescence substrate (Bio-Rad) and the G:BOX Chemi XX6 gel documentation system (Syngene).
Beesley J., Sivakumaran H., Moradi Marjaneh M., Lima L.G., Hillman K.M., Kaufmann S., Tuano N., Hussein N., Ham S., Mukhopadhyay P., Kazakoff S., Lee J.S., Michailidou K., Barnes D.R., Antoniou A.C., Fachal L., Dunning A.M., Easton D.F., Waddell N., Rosenbluh J., Möller A., Chenevix-Trench G., French J.D, & Edwards S.L. (2020). Chromatin interactome mapping at 139 independent breast cancer risk signals. Genome Biology, 21, 8.
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2

Quantification of CB2 Receptor Protein

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Samples of plantar paw skin, sciatic nerve and lumbar spinal cord were dissected from euthanized mice, frozen on dry ice, and stored at –80°C until time of processing. Samples of skin and nerve were pooled from both sides of one mouse. On the day of processing, samples were homogenized in a bullet blender in single-detergent lysis buffer (50 mM Tris-HCl, pH 8.0 with 1% Triton X-100, 150 mM NaCl, 0.02% Na azide, 100 μg/ml PMSF, and 1 μg/ml protease inhibitor mixture, Sigma), and the supernatant was obtained after centrifugation at 800 ×g for 10 min. The supernatant was concentrated using an Amicon Ultra-0.5 centrifugal filter (Millipore Corporation, Billerica, MA). Western blot analysis for CB2 receptor protein was performed on 25 μg of protein/sample as previously described [34 (link)] using rabbit anti-CB2 receptor antibody (dilution 1:500, Cayman). Actin immunoreactivity (rabbit anti-actin antibody, 1:2000, Sigma) within each sample was then quantified as a loading control. The density of immunoreactive bands was quantified using ImageJ (version 1.48r 18, NIH). Data for CB2 immunoreactivity were normalized to the amount of actin immunoreactivity within each sample (i.e., density of CB2 /density of actin). The quality of the material used in the analysis is provided in a supplemental figure.
Khasabova Iryna A., Yao X., Paz J., Lewandowski Cutler T., Lindberg Amy E., Coicou L., Burlakova N., Simone Don A, & Seybold Virginia S. (2014). JZL184 is anti-hyperalgesic in a murine model of cisplatin-induced peripheral neuropathy. Pharmacological research : the official journal of the Italian Pharmacological Society, 0, 67-75.
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3

Western Blot Analysis of HCMV Proteins

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Western blot was performed on triplicate infections carried out in confluent HFF in 6-well plates at the indicated MOI and treatment conditions. Sonicated whole-cell extracts in lysis buffer containing phosphatase and protease inhibitors were prepared as previously described (44 (link)). The gel-loading buffer contained 2% SDS and 100 mM beta-mercaptoethanol. The proteins were fractionated on freshly made 8% SDS-PAGE Tris-glycine gels prior to transfer to Amersham Protran 0.45-μm nitrocellulose membranes (GE Healthcare Life Sciences 10600002). HCMV IE1-72 and IE2-86 were detected by murine monoclonal antibody MAB810 (EMD Millipore, 1:1,000 dilution). Host actin was detected with polyclonal rabbit anti-actin antibody (Sigma-Aldrich, A2066, 1:4,000 dilution). Primary antibodies were detected with peroxidase AffiniPure F(ab’)2 fragment goat anti mouse IgG (Jackson ImmunoResearch, 115-036-006) at 1:40,000 dilution and goat anti-rabbit IgG (whole molecule)–peroxidase antibody (Sigma-Aldrich, A0545) at 1:40,000 dilution. Blots were imaged and analyzed using the iBright FL1500 Imaging System (Invitrogen).
Forte E., Li M., Ayaloglu Butun F., Hu Q., Borst E.M., Schipma M.J., Piunti A., Shilatifard A., Terhune S.S., Abecassis M., Meier J.L, & Hummel M. (2023). Critical Role for the Human Cytomegalovirus Major Immediate Early Proteins in Recruitment of RNA Polymerase II and H3K27Ac To an Enhancer-Like Element in OriLyt. Microbiology Spectrum, 11(1), e03144-22.
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4

Innate Immune Pathway Activation Assay

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Polyinosinic-polycytidylic acid (poly I:C; P1530) and rabbit anti-actin antibody were purchased from Sigma (St. Louis, MO). FBS was obtained from BIOWEST (Nuaille, France). Human type I interferon neutralizing antibody mixture (39000-1) was purchased from PBL Assay Science (Piscataway, NJ, USA). Small interfering RNA (siRNA) against MDA5 (SI03649037) and validated non-silencing negative control siRNA (1027281) were from Qiagen (Hilden, Germany). Lipofectamine RNAi-MAX reagent and M-MLV reverse transcriptase were from ThermoFisher (Carlsbad, CA, USA). The NucleoSpin RNA isolation kit was purchased from Macherey-Nagel (Duren, Germany). SsoAdvanced Universal SYBR Green Supermix was from Bio-Rad (Hercules, CA, USA). Primer oligo(dT)18 and oligonucleotide primers for polymerase chain reaction (PCR) were synthesized by Greiner Japan (Atsugi, Japan). A rabbit polyclonal antibody against MDA5 was from Immuno-Biological Laboratories (Fujioka, Japan). An enzyme-linked immunosorbent assay (ELISA) kit for CXCL10 was from R & D Systems (Minneapolis, MN, USA).
Saruga T., Imaizumi T., Kawaguchi S., Seya K., Matsumiya T., Sasaki E., Sasaki N., Uesato R, & Ishibashi Y. (2021). Role of MDA5 in regulating CXCL10 expression induced by TLR3 signaling in human rheumatoid fibroblast-like synoviocytes. Molecular Biology Reports, 48(1), 425-433.
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5

Autophagy regulation by eNOS signaling

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Tunicamycin, chloroquine, N-acetyl cysteine (NAC), DETA-NONOate, mammalian cell lysis kit, Rabbit anti-actin antibody, DMEM:F12 medium, Mission esiRNA for SQSTM1, and eNOS siRNA were purchased from Sigma-Aldrich. Glycan Differentiation kit, WST-1 cell viability kit, and X-tremeGENE HP DNA Transfection reagent were obtained from Roche. ROS detection kit, gold antifade mounting medium, NuPAGE Novex 4-12% Bis-Tris gels, goat anti-rabbit IgG-alexa 488, Goat-anti-rabbit IgG-Alexa Fluor 568, First Strand cDNA synthesis kit, Fetal Bovine Serum were from Invitrogen. JC1 mitochondrial membrane potential assay kit was from Cayman. Apodirect TUNEL assay kit was purchased from Millipore. Ultralow attachment tissue culture plate was from Nunc. TurboFect siRNA Transfection Reagent, DAPI, and HRP chemiluminescence substrate were from Thermo Scientific. Anti-ubiquitin antibody (clone P4D1) was from Biolegend. Anti rabbit antibodies against LC3, p62, and calnexin, Rabbit mAb phosphor-p70 S6 Kinase (Thr 389), mouse anti-eNOS mAb were obtained from Cell signaling Technology. pcDNA3-eNOS-GFP was obtained from Addgene. LC3-GFP and p62-EGFP constructs were kind gifts from Dr. Beth Levine (Columbia University, NY) and Dr. Terje Johansen (University of Tromso, Norway), respectively.
Guha P., Kaptan E., Gade P., Kalvakolanu D.V, & Ahmed H. (2017). Tunicamycin induced endoplasmic reticulum stress promotes apoptosis of prostate cancer cells by activating mTORC1. Oncotarget, 8(40), 68191-68207.
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6

Analysis of FAK and Akt Signaling

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KS1767 cells were seeded (1 × 106) into 6 well plates and grown to 80% confluence. Cells were incubated with each peptide (100 µM) for 10, 30 and 60 min, followed by protein extraction in 50 mM Tris, 150 mM NaCl, 1% NP-40, phosphatase/protease inhibitors (Roche). Protein extracts were resolved by SDS-PAGE, transferred to nitrocellulose membranes and decorated with standard protocols. The following primary antibodies (all Cell Signaling) were used at a 1:1,000 dilution: rabbit polyclonal anti-Fak (#3285), rabbit monoclonal anti-phosphorylated Fak (Tyr397, #8556), mouse monoclonal anti-Akt (2H10), rabbit monoclonal anti-phosphorylated Akt (Ser473, D9E), rabbit monoclonal anti-phosphorylated p44/p42 (Thr202/Tyr204), and mouse monoclonal anti-Erk1/2 (p44/p42) (3A7). The rabbit anti-actin antibody (Sigma) was used at a 1:2,000 dilution.
Staquicini D.I., Rangel R., Guzman-Rojas L., Staquicini F.I., Dobroff A.S., Tarleton C.A., Ozbun M.A., Kolonin M.G., Gelovani J.G., Marchiò S., Sidman R.L., Hajjar K.A., Arap W, & Pasqualini R. (2017). Intracellular targeting of annexin A2 inhibits tumor cell adhesion, migration, and in vivo grafting. Scientific Reports, 7, 4243.
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7

Immunoblot Analysis of Cell Signaling

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Protein extracts from islets and MIN6 transfected cells were prepared in lysis buffer (50 mmol/l Tris pH 7.5, 5 mmol/l EDTA, 150 mmol/l NaCl, 1% Triton X-100, 10 mmol/l sodium phosphate) containing fresh protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland). Proteins were separated by 8% SDS-PAGE and transferred to nitrocellulose membranes. Immunoblots were performed using the following antibodies: rabbit monoclonal anti-PTP1B antibody (Novus Biologicals, Littleton, CO, USA), rabbit anti-STAT3 antibody, rabbit anti-pSTAT3Tyr705 antibody, rabbit anti-AKT IgG, rabbit anti-pAKTThr308 antibody, rabbit anti-ERK1/2 (p44/42 MAPK) antibody, rabbit anti-pERKThr202/Tyr204 antibody, rabbit anti-FOXO1 IgG, rabbit anti-p FOXO1Ser256 antibody (all of them from Cell Signaling) and mouse monoclonal anti-p53 antibody (ABCAM, Cambridge, UK). As a loading control, rabbit anti-actin antibody was used (Sigma Aldrich). The antibody dilution used was 1∶1000.
Immunoblots were developed with horseradish peroxidise-conjugated secondary antibodies (GE Healthcare Bio-Sciences Corp. Piscataway, NJ, USA) and visualized using enhanced chemiluminiscence (Thermo Fisher Scientific Inc. Waltham, MA, USA). Bands were detected by an ImageQuant LAS 4000 camera (GE Healthcare) and quantified by densitometry scanning with Image J software.
Fernandez-Ruiz R., Vieira E., Garcia-Roves P.M, & Gomis R. (2014). Protein Tyrosine Phosphatase-1B Modulates Pancreatic β-cell Mass. PLoS ONE, 9(2), e90344.
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8

Quantitative Protein Analysis by Western Blot

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Electrophoresis was performed on precast 4-15% polyacrylamide Tris-Glycine gels (Bio-Rad). Protein levels were normalized to 50 μg of protein per sample and resuspended with 4X laemmli sample buffer (Bio-Rad) before boiling (5 min at 95°C). Then, proteins were transferred onto a polyvinylidenedifluoride (PVDF) membrane (Bio-Rad). Membranes were blocked with Tris-Tween Buffered solution (TTBS: 10 mM Tris, 200 mM NaCl, 0.05% Tween 20, pH 7.4) containing 5% non-fat dry milk for 1 h at room temperature. For F-actin precipitation studies, blots were then incubated overnight at 4°C with a mouse anti-CaMKII beta antibody (0,5μg/mL; Thermofisher, 139800) and a rabbit anti-actin antibody (1/2000; Sigma, A2066). For variant expression analysis, blots were incubated with a rabbit anti-CaMKIIβ (1/1000; Abcam, ab34703). Primary antibodies were probed with a IRDye 800CW goat anti-rabbit IgG together with a IRDye 680RD goat-anti mouse IgG (Li-Cor Biosciences) during 1 h incubation at room temperature. After 3 washes with TTBS and one with PBS, membrane was scanned using the automated infrared imaging system Odyssey (Li-Cor Biosciences) according to manufacturer’s instructions.
Nicole O., Bell D.M., Leste-Lasserre T., Doat H., Guillemot F, & Pacary E. (2018). A novel role for CAMKIIβ in the regulation of cortical neuron migration: implications for neurodevelopmental disorders. Molecular psychiatry, 23(11), 2209-2226.
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9

Western Blot Analysis of PARP and Caspase-3

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PolyADP-ribose polymerase (PARP) and caspase-3 cleavage were assessed using a standard western blotting protocol [17] (link). Membranes were probed using polyclonal rabbit anti-PARP1 antibody (Abcam, Cambridge, Cambridgeshire, UK; 1∶1000), a rabbit monoclonal anti-ERCC1 antibody (Cell Signaling Technology, Danvers, MA, USA; 1∶1000) and a polyclonal rabbit anti-cleaved caspase-3 antibody (Cell Signaling Technology; 1∶1000). A rabbit anti-actin antibody (Sigma-Aldrich Co. Ltd; 1∶2000) was used as a loading control. Blots were scanned (Bio-Rad GS-800 Calibrated Densitometer; Bio-Rad, Hercules, CA, USA) and signal quantification was performed by densitometry using scanning analysis software (Quantity One; Bio-Rad).
Witney T.H., Fortt R.R, & Aboagye E.O. (2014). Preclinical Assessment of Carboplatin Treatment Efficacy in Lung Cancer by 18F-ICMT-11-Positron Emission Tomography. PLoS ONE, 9(3), e91694.
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10

Neurotrophic Properties of CX3CL1

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Recombinant human CX3CL1 was from Calbiochem/Merck (Nottingham, UK); D-serine was from Ascent Scientific (Bristol, UK); rabbit anti-phospho TrkB (Tyr 515) was from Abcam (Cambridge, UK), rabbit anti-phospo CREB (Ser 133) and rabbit anti-PARP were from Cell Signaling (Danvers, Ma, USA); secondary antibodies were from DAKO (Milan, Italy); culture media were from Invitrogen Life Technologies (San Giuliano Milanese, Italy); 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), in vitro Toxicology Assay Kit Lactic Dehydrogenase based (LDH assay), catalase, D-amino acid oxidase (DAAO), poly-L-lysine, rabbit anti-actin antibody and all the other reagents were from Sigma-Aldrich (Milan, Italy).
Lauro C., Catalano M., Di Paolo E., Chece G., de Costanzo I., Trettel F, & Limatola C. (2015). Fractalkine/CX3CL1 engages different neuroprotective responses upon selective glutamate receptor overactivation. Frontiers in Cellular Neuroscience, 8, 472.
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