For the preparation of cell lysates, 5 × 106 cells were harvested from Treg-inducing cell cultures and pelleted at 390 × g for 10 min at 4°C. Cell pellets were lysed with RIPA cell lysis buffer for 20 min on ice. Following the SDS-PAGE, protein samples were transferred to a PVDF membrane, and protein detection was performed in a chemiluminescence image station (MicroChemi, Biostep GmbH, Burkhardtsdorf, Germany). For the detection of histone acetylation in Tregs, anti-acetyl-Histone H3 and H4 Abs (Merck Millipore, Darmstadt, Germany) were used. As a loading control for total cell protein extracts, a monoclonal anti-mouse β-actin Ab (Sigma-Aldrich, Munich, Germany) was used.
Mouse anti β actin ab
Manufactured by Merck Group
Sourced in United States
The Mouse anti-β-actin Ab is a monoclonal antibody that specifically binds to the β-actin protein, which is a widely expressed and highly conserved cytoskeletal protein. This antibody can be used to detect and quantify the presence of β-actin in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
P erk1 2 t202 y204, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti phospho p44 42 mapk thr202 tyr204, by Cell Signaling Technology (1 mentions)
Anti phospho p38 mapk, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence plus kit, by GE Healthcare (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Rabbit anti phospho p38, by Cell Signaling Technology (1 mentions)
Rabbit anti p38, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho aurora a t288, by Cell Signaling Technology (1 mentions)
Igf ir, by Cell Signaling Technology (1 mentions)
Rabbit anti cyclin b1, by Santa Cruz Biotechnology (1 mentions)
Odyssey ir imaging system, by LI COR (1 mentions)
Anti mouse hrp, by Merck Group (1 mentions)
0.2 mm nitrocellulose membrane, by GE Healthcare (1 mentions)
C digit blot scanner, by LI COR (1 mentions)
Westernsure premium chemiluminescent substrate, by LI COR (1 mentions)
Anti sheep hrp, by Merck Group (1 mentions)
Image studiotm lite version 5, by LI COR (1 mentions)
9 protocols using mouse anti β actin ab
1
Characterization of Treg Histone Acetylation
2
Signaling Pathway Activation in Splenic B Cells
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Splenic B cells were incubated with mB-Fab′–anti-Ig without (−) or with streptavidin (sAg) at 4°C, washed, and warmed to 37°C for indicated times and lysed. Cell lysates were analyzed with SDS-PAGE and western blot and probed for pAkt (Ser473; Cell Signaling Technology), pERK1/2 (T202/Y204; Cell Signaling Technology), pBtk (pBtk, Y551; BD Biosciences), and pY (Millipore). Anti-mouse β-actin Ab (Sigma-Aldrich) was used for loading controls.
3
Quantitative Western Blot Analysis
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Western blot were performed as previously described (Etna et al., 2015 (link)). Briefly, 25 μg of total protein extracts were separated on 10% SDS-PAGE gel and blotted onto nitrocellulose membranes (Millipore). Blots were incubated with rabbit polyclonal anti-phospho-p38 MAPK (Thr180/Tyr182) (p-p38), rabbit polyclonal anti-phospho-p44/42 MAPK (Thr202/Tyr204) (p-p44/42) and rabbit polyclonal anti-phospho-NF-kB p65 (Ser536) (p-p65) Abs (Cell Signaling Technology, Danvers, MA). Detection was achieved using anti-rabbit horseradish peroxidase-conjugate secondary Ab (Santa Cruz Biotechnology), and visualized with Enhanced Chemiluminescence plus kit (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). A ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) instrument and ImageLab software (Bio-Rad) were used to reveal and analyze the chemiluminescence signal. For loading control, β-actin levels were quantified by using a mouse anti-β-actin Ab (Sigma Aldrich). Protein amount was normalized to the actin level by ImageLab software. Fold changes of each analyzed protein were calculated by dividing values obtained in infected conditions by those of the uninfected counterpart.
4
Protein Immunoblotting Assay Protocol
5
Immunohistochemical Evaluation of SLFN11 and Related Markers
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Continuous 3‐µm‐thick sections were used for IHC. The Abs used in this study were as follows: mouse anti‐SLFN11 Ab (D‐2, #sc‐515071, 1:50 dilution for IHC and 1:500 dilution for western blot; Santa Cruz Biotechnology), mouse anti‐phospho‐Histone H2AX (Ser139) (γH2AX) Ab (JBW301, #DAM1493341, 1:200 dilution for immunofluorescence and 1:500 dilution for western blot; Sigma‐Aldrich), mouse anti‐β‐actin Ab (#127M4866V, 1:20 000 dilution; Sigma‐Aldrich), mouse anti‐p53 Ab (NCL‐L‐p53‐DO7, 1:200 dilution; Leica Biosystems), mouse anti‐cytokeratin 5/6 Ab (M7237, 1:200 dilution; Dako), mouse anti‐GATA3 Ab (ACR405B, 1:200 dilution; BIOCARE), rabbit anti‐ programmed death ligand 1 (PD‐L1) Ab (ab205921, 1:400 dilution; Abcam). The IHC procedures for SLFN11 and other Abs were described previously.22 , 27 , 28 , 29 All staining was manually scored by two surgical pathologists (DT and NS) without the knowledge of clinical findings or patient outcome. Schlafen 11 was considered positive when at least 5% of the tumor cells were stained. P53, GATA3, and PD‐L1 were considered positive when at least 10%, 20%, and 1% of the tumor cells were stained, respectively.30 Cytokeratin 5/6 (CK5/6) was considered positive when a full layer of the tumor cells was stained. Consensus regarding interpretation was made when there were discordant results.
6
Western Blot Analysis of BATF3 Protein
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Proteins were separated by 10% SDS-PAGE and transferred to a 0.2 mm nitrocellulose membrane (GE Healthcare, Chicago, IL, USA). Western blotting was performed using sheep anti-BATF3 AB (R&D Systems) and mouse anti-β-actin AB (Sigma-Aldrich, St. Louis, MO, USA) at a dilution of 1:1000. Signal development was performed using HRP–conjugated secondary AB at a dilution of 1:10.000: anti-sheep HRP (Sigma-Aldrich) and anti-mouse HRP (Sigma-Aldrich). Protein bands were visualized by WesternSure® PREMIUM Chemiluminescent substrate (LI-COR, Lincoln, NE, USA) using the C-DiGit® Blot Scanner (LI-COR) and Image StudioTM Lite Version 5.2 software (LI-COR).
7
Detailed PCSK9 Protein Assay Protocol
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The following reagents were from commercial sources: goat anti-mouse LDLR, horseradish peroxidase (HRP)-conjugated anti-mouse PCSK9 antibodies (Ab), and PCSK9 enzyme-linked immunosorbent assay (ELISA) kit from R&D Systems; mouse anti-β-actin Ab, HRP-conjugated anti-goat Ab, and lipoprotein-depleted serum (LPDS) from Sigma; plasmid vectors pCIneo and pGL4.13-luc2 as well as the Luciferase Assay System from Promega; Western Lightning Chemiluminescence Reagent Plus from Perkin-Elmer; plasma lipids assay kits from BioVision; RNeasy extraction kit and Ni-NTA agarose from Qiagen; Superscript II RNase H– Reverse Transcriptase, PCR primers, HRP-conjugated anti-V5 tag Ab, and Lipofectamine Reagent Plus from Invitrogen; FastStart TaqMan ProbeMaster-Rox master mix, Universal Probe Library (UPL) fluorescent probes, and the Protease Inhibitor Cocktail (PIC) from Roche.
8
Immunostaining for DNA Damage Response
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Antibodies used include rabbit anti-KAP1 Ab (A300-274A, Bethyl Laboratories), rabbit anti-ATM Ab (A300-299A, Bethyl Laboratories), mouse anti-β-actin Ab (AC-15, Sigma), rabbit anti-p-S824-KAP1 Ab (A300-767A, Bethyl Laboratories), mouse anti-EA-D Ab (MAB8186, EMD), mouse anti-FLAG Ab (F3165, Sigma), goat anti-ATM Ab ((A300-136A, Bethyl Laboratories), rabbit anti-p-S139-H2AX (γH2AX) Ab (9718P, Cell Signaling Technology), normal rabbit IgG (sc-2027, Santa Cruz), normal goat IgG (sc-3887, Santa Cruz) and mouse anti-ZEBRA Ab (a gift from Professor Paul Farrell), HRP conjugated goat anti-mouse IgG (H+L) (AP308P, EMD Millipore), HRP conjugated goat anti-rabbit IgG (H+L)(AP307P, EMD Millipore), Fluorescein isothiocyanate (FITC) conjugated donkey anti-rabbit IgG (31568, Thermo Fisher), Phycoerythrin (PE) conjugated goat anti-mouse IgG (sc-3738, Santa Cruz), FITC conjugated goat anti-mouse IgG (F0257, Sigma), and Alexa Fluor 647 conjugated goat anti-rabbit IgG (A-21245, Thermo Fisher).
9
Immunoblotting with anti-CD98 and ATG7 Abs
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Unconjugated mouse anti-human CD98 (4F2hc, solute carrier family 3 member 2) Ab (#556074) and APC-conjugated goat anti-mouse Ig Ab (#550826) were purchased from BD BioSciences (San Jose, CA, USA). Mouse IgG1κ isotype control Ab (#400123-BL) was obtained from Biolegend (San Diego, CA, USA). Rabbit anti-human ATG7 Ab (#8558) was purchased from Cell Signaling Technology (Danvers, MA, USA), and mouse anti-β-actin Ab (#A5441) was from Sigma-Aldrich. IRDye 800CW donkey anti-rabbit (#925-32213) and IRDye 680RD donkey anti-mouse (#925-68072) secondary antibodies were purchased from LI-COR (Lincoln, NE, USA).
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