HepG2 Cells were lysed in 2× SDS sample buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM DTT, and 0.05% bromophenol blue) and sonicated for 10 s to shear DNA. The whole-cell extracts were then electrophoresed through 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc) with a Mini PROTEANII transfer apparatus (Bio-Rad). The membranes were blocked and probed in TBS-T buffer (1× Tris-HCl 50 mM pH7.6, NaCl 150 mM, 0.2% Tween-20) containing 5% non-fat milk. Primary antibodies (Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370, Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb #4668, Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb #4511, Cell Signaling Technology, Inc.) at 1:1000 in TBS-T were incubated with the membrane overnight at 4°C. β-actin was used as an internal control for normalization and detected with a monoclonal actin antibody diluted at 1:2500 (Sigma, St. Louis, MO). After washing with TBS-T, the membranes were then further incubated with horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG (1:5000; Sigma), The immunoreactive bands were visualized by enhanced chemiluminescence reagents (Perkin-Elmer Life Science Products, Boston, MA), and densitometry scanning was performed with the densitometer (Molecular Dynamics).
Actin monoclonal antibody
Manufactured by Merck Group
Sourced in United States
The ACTIN monoclonal antibody is a laboratory reagent produced by Merck Group. It is designed for the detection and localization of actin, a key structural protein found in all eukaryotic cells. The antibody can be used in various immunological techniques, such as immunofluorescence microscopy, Western blotting, and ELISA, to study the distribution and expression of actin within cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (4 mentions)
Enhanced chemiluminescence detection kit, by Cytiva (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (3 mentions)
Bradford method, by Bio-Rad (3 mentions)
Horseradish peroxidase conjugated anti mouse igg and anti rabbit igg, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Sox30 rabbit polyclonal antibody, by Santa Cruz Biotechnology (2 mentions)
Tc2n rabbit polyclonal antibody, by Abcam (2 mentions)
P21 mouse monoclonal antibody, by Santa Cruz Biotechnology (2 mentions)
Densitometer, by GE Healthcare (1 mentions)
Pabpn1, by Abcam (1 mentions)
Dsred, by Abcam (1 mentions)
Immobilon western chemiluminescent hrp substrate kit, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
E2f1 antibody, by Cell Signaling Technology (1 mentions)
Wnt5a, by GeneTex (1 mentions)
Fibronectin, by GeneTex (1 mentions)
Twist1, by GeneTex (1 mentions)
Snail, by GeneTex (1 mentions)
Protease inhibitors cocktail, by Merck Group (1 mentions)
21 protocols using actin monoclonal antibody
1
Western Blot Analysis of Phosphorylated MAPKs
2
Western Blot Analysis of Protein Expression
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Cell lysis was performed using 2x SDS sample buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, 50 mM DTT, and 0.05% bromophenol blue). Cell lysates were sonicated for 10 s to shear DNA. Cell extracts were then electrophoresed through 10% SDS-polyacrylamide gels and transferred to 0.2 µm nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA) with a Miniprotein II transferring apparatus (Bio-Rad). The non-specific interaction of membrane was blocked with 5% non-fat milk and probed in TBS-T buffer (1x TBS buffer, 0.2% Tween 20) containing. Monoclonal rabbit anti-AR (1:5000), was used to detect the androgen receptor (Abcam #133273), Glucocorticoid Receptor (D8H2) XP® Rabbit mAb #3660, ERα Antibody (F-10): sc-8002, BRD4 (E2A7X) Rabbit mAb #13440, Brd2 (D89B4) Rabbit mAb #5848, PARP (46D11) Rabbit mAb #9532, Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb #8173 and a monoclonal actin antibody diluted 1:2500 (Sigma, St. Louis, MO) was used to detect β-actin as the internal control to normalize protein loading. The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG (1: 5,000; Sigma). Enhanced chemiluminescence reagents (Perkin-Elmer Life Science Products, Boston, MA) were used to visualize the immunoreactive bands and the densities of protein bands were scanned analyzed using ImageJ software from the NIH.
3
PABPN1 Protein Expression Analysis
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Total proteins were extracted in SUB buffer at different time points (containing 8M urea, 2% mercaptoethanol and 0.5% SDS), and electrophoresed in 12% SDS-PAGE gels. Proteins were blotted into nitrocellulose membrane and probed with specific antibodies against PABPN1 (1:2000) (Abcam, USA). Parallel samples were probed using monoclonal actin antibody (Chemicon International, USA) to confirm the equal loading of lysates between lanes. After incubation with specific secondary HRP-conjugated antibodies, the membranes were revealed using the western blot chemiluminescence reagent plus kit (NEN Life Sciences Products, Boston, MA, USA).
Quantification of the band intensity was performed using the ImageJ software. Values were normalized by β-actin. Quantification of PABPN1 protein level was determined by western blot using the ImageJ software, normalized to actin and presented as fold change relative to the level of PABPN1 in untransfected HEK293T cells.
Quantification of the band intensity was performed using the ImageJ software. Values were normalized by β-actin. Quantification of PABPN1 protein level was determined by western blot using the ImageJ software, normalized to actin and presented as fold change relative to the level of PABPN1 in untransfected HEK293T cells.
4
Quantitative Protein Expression Analysis
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Total proteins were extracted in sodium dodecyl sulfate utilizing buffer (SUB) at different time points (containing 8M urea, 2% β-mercaptoethanol, and 0.5% SDS) and electrophoresed in 12% SDS-PAGE gels. Proteins were blotted on nitrocellulose membranes and probed with specific antibodies against PABPN1 (1:2,000) (Abcam, USA), Dsred (1:20) (Biovision, USA), and myosin (1:200) (DSHB, Iowa City, IA, USA). Parallel samples were probed using monoclonal actin antibody (Chemicon International, USA) to confirm the equal loading of lysates between lanes. After incubation with specific secondary horseradish peroxidase (HRP)-conjugated antibodies, the membranes were revealed using the western blot chemiluminescence reagent plus kit (NEN Life Sciences Products, Boston, MA, USA).
5
Protein Extraction and Western Blot Analysis of Colon Cancer Samples
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The colon cancer samples were lysed with cell lysis buffer 20 mM Tris-HCl (pH 7.5), 20 mM β-glycerophosphate, 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM PMSF, 10 mM NaF, 10 µg/ml leupeptin, 2 µg/ml aprotinin,1% Triton X-100 and 1 mM EDTA]. Protein concentration was determined using the bicinchoninic acid method and 80 µg of total protein was used for western blotting. Protein lysates were separated using 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked for 1 h in 5% BSA and incubated with primary antibodies overnight at 4°C, washed, and incubated with secondary antibodies (1:5,000) for 1 h. The membranes were then washed and the protein bands were visualized using the Immobilon Western Chemiluminescent HRP Substrate Kit (EMD Millipore).
Western blotting was performed with the following primary antibodies: actin monoclonal antibody (1:5,000; cat. no. A5441; Sigma-Aldrich; Merck KGaA), Twist1 (1:1,000; cat. no. 46702; Cell Signaling Technology, Inc.) and COL11A1 (1:1,000; cat. no. 42818; Cell Signaling Technology, Inc.). The horseradish peroxidase-conjugated secondary goat anti-rabbit and goat anti-mouse (1:5,000; cat. nos. 31466 and A16078; Thermo Fisher Scientific, Inc.). densitometry was performed using Quantity One software (Bio-Rad Laboratories, Inc.).
Western blotting was performed with the following primary antibodies: actin monoclonal antibody (1:5,000; cat. no. A5441; Sigma-Aldrich; Merck KGaA), Twist1 (1:1,000; cat. no. 46702; Cell Signaling Technology, Inc.) and COL11A1 (1:1,000; cat. no. 42818; Cell Signaling Technology, Inc.). The horseradish peroxidase-conjugated secondary goat anti-rabbit and goat anti-mouse (1:5,000; cat. nos. 31466 and A16078; Thermo Fisher Scientific, Inc.). densitometry was performed using Quantity One software (Bio-Rad Laboratories, Inc.).
6
Comprehensive Protein Analysis Techniques
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Western blot analysis and immunohistochemical (fluorescence) staining were conducted as previously described.14 (link), 15 (link) The primary antibodies used in this study were actin monoclonal antibody (1:5000, Merck, Darmstadt, Germany), E2F1 antibody (1:1000, Cell Signaling Technology, Beverly MA, USA), GFP monoclonal antibody (1:200, Upstate, Lake Placid, NY, USA), FITC-conjugated anti-rabbit, rhodamine-conjugated anti-mouse, alkaline phosphatase–conjugated anti-rabbit antibody (1:500, Jackson ImmunoResearch Laboratories, West Grove, PA, USA), SPZ1, TWIST1, AKT, Ki67, HIF-1, Snail, Slug, Wnt5a, ZEB1, BMI1, fibronectin, E-cadherin rabbit polyclonal antibody (1:1000; GeneTex, Irvine, CA, USA). ERK1/2, PI3K monoclonal antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). CD133, CD44, N-cadherin, vimentin rabbit polyclonal antibody (1:500, Abcam, Cambridge, MA, USA) All of the experiments were repeated at least three times.
7
Western Blot Analysis of One-Carbon Metabolism Enzymes
8
Protein Isolation and Western Blot Analysis
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Proteins were isolated using lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, and 1% SDS) with a protease inhibitor cocktail (Sigma, St. Louis, MO, USA) on ice for 1 h and then centrifuged for 20 min at 13,000 ×g. The supernatant was collected, and protein concentrations were measured using the Bradford method (Bio-Rad, Hercules, CA, USA). Proteins were dissolved in the sample buffer and boiled for 5 min prior to loading onto an acrylamide gel. After SDSPAGE, the proteins were transferred to a polyvinylidene difluoride membrane and blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 60 min at room temperature. The membranes were incubated with the primary antibody for 2 h at room temperature. Equal lane loading was assessed using an actin monoclonal antibody (Sigma-Aldrich). After washing with TBST, the blots were incubated with a 1:5000 dilution of horseradish peroxidase-conjugated secondary antibody (Invitrogen, Grand Island, NY, USA) and washed again three times with TBST. The transferred proteins were visualized using an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech).
9
Western Blot Analysis of Apoptosis Markers
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Cells were lysed in RIPA buffer containing protease inhibitor cocktail (Sigma) and phosphatase inhibitors (Roche Applied Bioscience, Indianapolis, IN, USA). Cell lysates were subjected to SDS-PAGE followed by immunoblot with corresponding antibodies. Polyclonal antibodies against JNK1, JNK2, pJNK, Bcl-XL, Bcl-2, BAX, phospho threonine and COX IV were from Cell Signaling Technology (Beverly, MA, USA; all used at 1:1000). Actin monoclonal antibody (1:5000) was from Sigma. ALDH1L1 and Bid were detected using in-house polyclonal antibodies (1:20 000) generated against full-length proteins. Rabbit polyclonal phospho-Bid-specific (pT59) antibody was generated by AnaSpec (Freemont, CA, USA) against ELQ(pT)DGNRSSHSRLG peptide (1:3000). In all experiments, a Hybond TM-ECL nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburg, PA, USA) and Pierce ECL detection kit (Thermo Scientific, Waltham, MA, USA) were used.
10
Western Blot and IHC Analysis of NF-κB Pathway
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WB was performed as previously described [17 (link)]. ACTIN was used as a loading control. The following primary antibodies were used: Tac2-N rabbit polyclonal antibody (1:500; Abcam), p65 rabbit polyclonal antibody (1:1000; Abcam), p-p65S536 rabbit polyclonal antibody (1:1000; Abcam), p50 rabbit polyclonal antibody (1:1000; Abcam), p-p50S337 rabbit polyclonal antibody (1:1000; Abcam), IκBα rabbit polyclonal antibody (1:1000; Abcam), p- IκBαS32 rabbit polyclonal antibody (1:1000; Abcam), p- IκBαS36 rabbit polyclonal antibody (1:1000; Abcam), beta-Tubulin rabbit polyclonal antibody (1:500, Bioss), Lamin A/C rabbit polyclonal antibody (1:500, Bioss) and ACTIN monoclonal antibody (1:2000; Sigma).
IHC staining was performed using a rabbit monoclonal antibody against Vimentin (1:500; Abcam) as described previously [12 (link)]. The vimentin staining was used to identify tumor cells in mice lungs and was also performed on paraffin-embedded tissues. The number and the size of metastatic foci were evaluated and quantified using vimentin staining.
IHC staining was performed using a rabbit monoclonal antibody against Vimentin (1:500; Abcam) as described previously [12 (link)]. The vimentin staining was used to identify tumor cells in mice lungs and was also performed on paraffin-embedded tissues. The number and the size of metastatic foci were evaluated and quantified using vimentin staining.
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