Antibodies against the following proteins were used for immunoblot, listed by manufacturer: Atlas antibodies: TASOR (HPA006735). Bethyl: BBX (A303-151A), HLTF (A300-230A), RALY (A302-070A), ZNF512B (A303-234A). Cell Signaling Technology: SMN1/2 (2F1). Novus: ESCO2 (NB100-87021). Origene: UNG2 (2C12). Proteintech: Vpr (51143-I-AP). Santa Cruz: CCNB1 (SC-245), ZGPAT (SC-515524). Sigma: β-actin (AC74). Abcam: p24 (ab9071), VCP (ab11433). The following secondary antibodies were used: goat anti-mouse-HRP and goat anti-rabbit-HRP (immunoblot, Jackson ImmunoResearch, West Grove, PA). Blots were immersed in chemiluminescent substrate (SuperSignal West Pico or Dura, Thermo Fisher Scientific), and signal was visualized using X-ray film or the iBright CL1000 imaging system (Thermo Fisher Scientific).
β actin ac 74
Manufactured by Merck Group
Sourced in United States
β-actin (AC-74) is a laboratory reagent produced by Merck Group. It is a monoclonal antibody that specifically binds to the cytoskeletal protein β-actin. β-actin is a ubiquitous and highly conserved protein found in all eukaryotic cells and is involved in various cellular processes such as cell motility, structure, and division.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (3 mentions)
Chemidoc touch, by Bio-Rad (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Noxa 114c307.1, by Thermo Fisher Scientific (2 mentions)
Mcl 1, by Cell Signaling Technology (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Gapdh 14c10, by Cell Signaling Technology (2 mentions)
Ha 3f10, by Roche (2 mentions)
Secondary antibodies coupled to horseradish peroxidase hrp, by Agilent Technologies (2 mentions)
Flag m2, by Merck Group (2 mentions)
Hpv16 e6 e6 6f4, by Euromedex (2 mentions)
Igg anti mouse igg h l hrpo, by Dianova (2 mentions)
Bca assay, by Bio-Rad (2 mentions)
Hif 1α, by BD (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ibright cl1000 imaging system, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
P24 ab9071, by Abcam (1 mentions)
Goat anti mouse hrp, by Jackson ImmunoResearch (1 mentions)
Goat anti rabbit hrp, by Jackson ImmunoResearch (1 mentions)
29 protocols using β actin ac 74
1
Immunoblotting Antibody Panel for Diverse Proteins
2
Phagosome Protein Analysis by Western Blot
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Purified phagosomes (20 µg) or total cell lysates (60 µg) from BMDCs were run on SDS-PAGE with 4–12% gradient gel and then transferred. The membranes were blocked with 5% dried milk and incubated with primary antibodies overnight. After washing with PBS with Tween 20, secondary antibodies were incubated with the membranes for 1.5 h. The bands were visualized by chemical composition using ChemiDoc Touch (Bio-Rad). The gray area ratio of different bands was calculated by software from ChemiDoc Touch (Bio-Rad). The following antibodies were used: PPT1 (N1C3; GeneTex), catS (E-3; Santa Cruz Biotechnology), ATP6V1A (ab137574; Abcam), and β-actin (AC74; Sigma-Aldrich).
3
Immunoblotting Analysis of Apoptosis and Signaling Pathways
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Antibodies used were: cleaved caspase-3 (#9661), cleaved poly (ADP-ribose) polymerase (#9541), AKT-total (#4691), phospho-AKT(Ser473) (#4060), JAK2 (#9945S), phospho-SAPK/JNK (Thr183/Tyr185) (#6251S), STAT3 (#9139S), phospho-STAT3 (Tyr705) (9145S) and phospho-STAT3 (Ser 727), E-cadherin (#4065) (9134) (Cell Signaling, Beverly, MA); Flag M2 (F3165), Vimentin (V6630), DLEC1 (HPA019077) and β-actin (AC-74) (Sigma-Aldrich, St. Louis, MO); anti-mouse Ig G-HRP (P0161), anti-rabbit Ig G-HRP (P0448) (Dako ,Glostrup, Denmark); Twist (sc-15393; Santa Cruz, CA, USA); a-tubulin (Lab Vision Corporation, Fremont, CA); V5-Tag (MCA1360; AbD Serotec, Raleigh, NC).
Human IL-6 (PF01229) (Peprotech, Rocky Hill, NJ) was used. Western blot and IP experiments were performed according to previous protocols 48 (link), 49 (link).
Human IL-6 (PF01229) (Peprotech, Rocky Hill, NJ) was used. Western blot and IP experiments were performed according to previous protocols 48 (link), 49 (link).
4
Protein Extraction and Western Blot Analysis
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Proteins from cultured cells were extracted by lysis in RIPA buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5% sodium deoxycholic acid, 0.5% NP-40, 0.1% SDS) supplemented with a protease inhibitor cocktail (Complete; Roche Molecular, Neuilly sur Seine, France). They were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon, Millipore, Billerica, CA) by electroblot at 4°C for 90 minutes at 90 V or overnight at 45 V. The antibodies used for Western blot analysis were mouse monoclonal antibodies directed against the human TLR3 (clone 512505, ref. MAB1487, R&D Systems, Minneapolis, MN), HIF-1α (610958; BD Biosciences, Bedford, MA), β-Actin (AC-74; Sigma Aldrich) and α-Tubulin (B-5-1-2; Sigma Aldrich). Blotted membranes were incubated with a secondary peroxidase-conjugated antibody, and chemiluminescent detection was done using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, CA). The acquisition was performed with ImageQuant LAS 4000 mini biomolecular imager (GE Healthcare Bio-Sciences AB) and specific protein bands were quantified using ImageQuant TL software (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
5
Antibody Acquisition and Small Molecule Procurement
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Antibodies were purchased as follows: BIM (C34C5), BCL-XL (54H6), CREB (48H2) and cleaved caspase-3 (5A1E) from Cell Signaling Technology (Danvers, MA, USA); NOXA (114C307.1) from Thermo Fisher Scientific (Waltham, MA, USA); ATF-3 (C-19) and MCL-1 (S-19) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); KLF4 (56CT5.1.6) from Bioss, Inc. (Woburn, MA, USA); α-tubulin (DM1A) and β-actin (AC-74) from Sigma-Aldrich (Tokyo, Japan). APTO-253 was purchased from MedChemExpress, LLC (Monmouth Junction, NJ, USA). Q-VD-OPh, Doxorubicin, etoposide, SP600125, SB203580, PD184352, and U0126 were purchased from Calbiochem (San Diego, CA, USA). Hydroxychloroquine (HCQ), Necrostatin-1 and Ferrostatin-1 were purchased from Cayman Chemical (Ann Arbor, MI, USA).
6
Western Blot Analysis of Apoptosis Regulators
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Proteins were extracted with CelLyticTM M Cell Lysis Reagent (Sigma‐Aldrich, Dorset, UK) supplied with protease and phosphatase inhibitor cocktails (Sigma‐Aldrich, Dorset, UK). Proteins were mixed with NuPAGE LDS Sample Buffer (Invitrogen, Carlsbad, CA) and boiled for 5 min before analysis by western blot analysis. Proteins were subjected to 4–20% NuPAGE gels (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membrane (Sigma‐Aldrich, Dorset, UK) at 20 V for 1 hr by semidry transfer. PVDF membrane was blocked with 5% nonfat milk in TBST for 1 hr and then incubated with primary antibodies overnight at 4°C against the following targets: Bcl‐2 (100), RelA (NF‐κB p65, F‐6), Bcl‐XL (H‐5), Bax (2D2), Survivin (D‐8), and LDH (H‐10) (Santa Cruz Biotechnology, Inc., Dallas, TX); β‐actin (AC‐74) (Sigma‐Aldrich, Dorset, UK); p‐RelA (phospho‐NF‐κB p65‐Ser536), p‐STAT3 (phosphor‐STAT3‐Tyr705), p‐AKT, AKT, Mcl‐1, and Rb antibodies (Cell Signaling Technology‐New England Biolabs, Hitchin, UK). Bound antibodies were detected using appropriate horseshoeradish peroxidase‐conjugated secondary antibodies and visualized by GeneSnap (SynGene, Cambridge, UK) after adding ECL plus (GE Healthcare Life Science, Hatfield, UK).
7
Protein Detection and Expression
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For protein detection, western blotting and immunofluorescence staining were used. For protein expression analysis, cells were harvested in SDS-Triton lysis buffer. Protein concentration was measured using Bio-Rad DC Protein assay and protein detection using Pierce ECL Western blotting substrate (Thermo Scientific). Antibodies used were: HIF-1α (610959, BD Transduction Laboratories), phospho-H2A.X (Ser139) (2577, Cell Signaling), H2A.X (2595, Cell Signaling), GAPDH (5G4-6C5, HyTest), and β-actin (Ac-74, Sigma-Aldrich).
For immunofluorescence staining, cells were fixed with ice cold methanol. The DNA double strand breaks were visualized using antibody against phosphorylated H2A.X (γH2A.X) (2577, Cell Signaling). The nuclei were stained using Hoechst 33342 (Invitrogen). Cells were imaged with Olympus BX60 (40X). The intensity of the phosphorylated H2A.X staining per cell (minimum 4 optical fields yielding minimum 150 cells per condition) was measured using ImageJ software (NIH, USA). Experiments were done as parallel treatments and each experiment was repeated at least three times.
For immunofluorescence staining, cells were fixed with ice cold methanol. The DNA double strand breaks were visualized using antibody against phosphorylated H2A.X (γH2A.X) (2577, Cell Signaling). The nuclei were stained using Hoechst 33342 (Invitrogen). Cells were imaged with Olympus BX60 (40X). The intensity of the phosphorylated H2A.X staining per cell (minimum 4 optical fields yielding minimum 150 cells per condition) was measured using ImageJ software (NIH, USA). Experiments were done as parallel treatments and each experiment was repeated at least three times.
8
Immunoblotting and Flow Cytometry Protocols
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Western blots, prepared by standard procedures, were probed using the following antibodies: c-Myc (N-262), Max (C-17) and Mnt (M-132) all from Santa Cruz Biotechnology (Dallas, TX, USA), or c-Myc (D84C12) from Cell Signaling Technology (Danvers, MA, USA) and Mnt (A303-626A) from Bethyl Laboratories (Montgomery, TX, USA) and β-actin (AC-74, Sigma-Aldrich, St. Louis, MO, USA). Blots in Figure 1b were imaged on a ChemiDoc Touch (Bio-Rad, Hercules, CA, USA) and analysed using Image Lab software (Bio-Rad). Monoclonal antibodies used for flow cytometry, produced and labelled with FITC, PE or APC in house, included the following: RB6-8C5, anti-Gr1; MI/70, anti-Mac1; YTA3.2.1, anti-CD4; 53.6.7.2, anti-CD8; Ter119, anti-erythroid marker; ID3, anti-CD19; RA3-6B2, anti-CD45R-B220; 5.1, anti-IgM; 11-26C, anti-IgD; 145-2C11, anti-CD3; E13.161.7, anti-Sca-1; 57, anti-CD43; T329.1, anti-Thy1; A20.1 anti-Ly5.1.
9
Cytotoxicity Evaluation of Chemotherapeutics
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Cytosine β-D-arabinofuranoside hydrochloride (Ara-C HCl), Daunorubicin hydrochloride (Dnr HCl) and Methyl thiazolyldiphenyl-tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St Louis, MO, USA). Arsenic trioxide (ATO; Arsenox) was purchased from Intas Pharmaceuticals (Ahmedabad, India). Brusatol (NSC 172924-T/1) was provided by Developmental Therapeutics Program, National Cancer Institute (NCI-DTP, Bethesda, USA). All cell culture reagents were obtained from Thermo Scientific (Waltham, MA, USA). Rabbit polyclonal anti Nrf2 (H300, 1:200) was purchased from Santa Cruz Technologies (CA, USA). Rabbit monoclonal anti-Nrf2 (D1Z9C, 1:6400) for use in flowcytometry experiments, was purchased from Cell Signalling Technologies (Danvers, MA, USA), β Actin (AC-74, 1:5000) from Sigma-Aldrich (St. Louis, MO), rabbit polyclonal anti-KEAP1 from Cuz Bio (CSB-PA012147LA01HU) and horseradish peroxidase-conjugated goat anti-Rabbit (1:4000), goat anti-mouse IgG (1:20,000) secondary antibodies from Thermo Scientific (Waltham, MA, USA).
10
Antibody Sources and Plasmid Constructs
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The following antibodies were purchased from commercial sources: HA antibody (Covance), GFP antibody (D5.1; Cell Signaling Technology), β-actin (AC-74; Sigma-Aldrich), VP16 (ab4808-100; Abcam), RAS (Ab-3, OP40; EMD Millipore), ERK1 (sc-94, K-23; Santa Cruz Biotechnology, Inc.), and β-tubulin (E7-s; Developmental Studies Hybridoma Bank). GFP-agarose beads (D153-8) were from MBL International. pCXN2-HA-human GPR31 was obtained from J. Miyazaki (RIKEN Center for Developmental Biology, Kobe, Japan; Niwa et al., 1991 (link)). Human GPR31 was inserted into pCGN-HA vector and pEGFP-N1 by standard PCR cloning. Human β2AR was inserted into pEGFP-N1 (β2AR-1) and was also obtained in S65TGFP vector (β2AR-2) from L. Barak (Duke University, Durham, NC). The mouse formyl peptide receptor FPR-rs2 was received in pEGFP-N1 vector from P. Murphy (National Institute of Allergy and Infectious Diseases, Bethesda, MD). The peptide sequence from rat GABA receptor 1b, which contains the ER localization signal RSRR (LLEKENRELEKIIAEKEERVSELRHQLQSRQQLRSRR), was linked to the 3′ end of Flag-GPR31 (human) and cloned into pEGFP-N1. KRAS4B was cloned from pEGFP-C3-KRAS4B into ptdTomato-C1. FTI L-744,832 was purchased from Enzo Life Sciences, and GGTI-2418 was provided by S. Sebti (Moffitt Cancer Center, Tampa, FL).
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