100 μg of total protein from whole cell lysate or mitochondria fraction was immunoprecipitated. The extract was incubated for 16 h at 4 °C with anti-acetylated-lysine (Cell Signaling) followed by addition of protein G beads and incubated further for 6 h at 4 °C. The beads were centrifuged at 2000 rpm for 2 min and washed three times in PBS buffer. The beads were recovered by centrifugation and aliquots of pellets were analyzed by SDS–PAGE and immunoblotting. For Western blotting, anti-acetylated-lysine (Cell Signaling), anti-SIRT2, anti-SIRT3, anti-SIRT5, anti- SIRT6 (Cell Signaling), anti-COX IV (Cell Signaling), HRP conjugated anti-GAPDH (Cell Signaling), anti-APT5A (Abcam) and anti-PDH cocktail antibodies (Abcam) were used for detection.
Anti acetylated lysine
Manufactured by Cell Signaling Technology
Sourced in United States, China
Anti-acetylated-lysine is a laboratory product that detects the presence of acetylated lysine residues in proteins. It is a useful tool for studying post-translational modifications and protein regulation.
Automatically generated - may contain errors
Lab products found in correlation
Anti sirt3, by Cell Signaling Technology (6 mentions)
Anti flag, by Merck Group (5 mentions)
Anti β actin, by Merck Group (5 mentions)
Anti β actin, by Cell Signaling Technology (5 mentions)
Anti sirt1, by Cell Signaling Technology (5 mentions)
Anti nf κb p65, by Cell Signaling Technology (3 mentions)
Anti α tubulin, by Cell Signaling Technology (3 mentions)
Anti lamin b1, by Cell Signaling Technology (3 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Anti sirt1, by Merck Group (3 mentions)
Anti gapdh, by Cell Signaling Technology (3 mentions)
Anti cox 4, by Cell Signaling Technology (2 mentions)
Anti sirt6, by Cell Signaling Technology (2 mentions)
Lysis buffer, by Thermo Fisher Scientific (2 mentions)
Anti enos, by BD (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti p300, by Cell Signaling Technology (2 mentions)
Anti acetyl nfκb p65 lys310, by Cell Signaling Technology (2 mentions)
Nurd complex antibody sampler kit, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
43 protocols using anti acetylated lysine
1
Profiling Acetylated Mitochondrial Proteins
2
Endothelial Nitric Oxide Synthase Acetylation
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The PVAT tissue samples were ground to a fine powder in liquid nitrogen with a mortar, then lysis buffer (Thermo Fisher Scientific; Cat# 610297) was added to obtain the PVAT lysate. For performing the eNOS immunoprecipitation (IP), 1 mg PVAT lysate was incubated with 3 µg of the specific eNOS antibody (LifeSpan BioSciences; Cat# LS-C288673) for IP at 4 °C for 1 h. After that, the lysate was incubated with 50 μL Protein A/G Magnetic Beads at room-temperature for 1 h. The immunoprecipitates were boiled in a loading buffer for 5 min at 95 °C before being subjected to SDS/PAGE. Anti-acetylated lysine (Cell Signaling Technology; Cat# 9441) and the anti-eNOS (BD Transduction Laboratories; Cat# 610297) antibodies were used for immunoblotting, respectively.
3
Immunoblotting and Immunofluorescence of Acetylated Proteins
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Cells were washed twice with PBS and lysed in RIPA buffer. Protein concentrations were quantified using the Bradford reagent (Bio-Rad) according to the manufacturer's instructions. Samples with equal amounts of protein were separated by 4%–15% SDS-PAGE, then transferred to an Immobilon transfer membrane (Millipore) and immunoblotted with specific antibodies against E-cadherin, PCK2, PKM2, and Acetylated-Lysine (Ac-K2-100, #9814) from Cell Signaling, and GAPDH from Thermo Scientific. All of the immunoblots were visualized by enhanced chemiluminescene (Bio-Rad).
Immunofluoresence staining was performed to detect PCK2 expression and protein acetylation. Cells were plated in a 24-well multiwell glass-bottomed culture plate (MatTek), fixed in 4% formaldehyde in PBS for 15 min at room temperature, and then washed three times with PBS. After being blocked with PBS containing 5% goat serum and 0.3% Triton X-100 at room temperature for 1 h, the cells were incubated overnight with anti-PCK2 primary antibody (1:100, Cell Signaling) or anti- acetylated-Lysine (1:200, Cell Signaling #9441) at 4°C. The secondary antibody, Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Molecular Probes) was used at a 1:1000 dilution. Fluorescence was monitored by an inverted confocal laser microscope (Carl Zeiss).
Immunofluoresence staining was performed to detect PCK2 expression and protein acetylation. Cells were plated in a 24-well multiwell glass-bottomed culture plate (MatTek), fixed in 4% formaldehyde in PBS for 15 min at room temperature, and then washed three times with PBS. After being blocked with PBS containing 5% goat serum and 0.3% Triton X-100 at room temperature for 1 h, the cells were incubated overnight with anti-PCK2 primary antibody (1:100, Cell Signaling) or anti- acetylated-Lysine (1:200, Cell Signaling #9441) at 4°C. The secondary antibody, Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Molecular Probes) was used at a 1:1000 dilution. Fluorescence was monitored by an inverted confocal laser microscope (Carl Zeiss).
4
ADGRL4/ELTD1 Silencing and Panobinostat Treatment
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HUVECs underwent ADGRL4/ELTD1 silencing as described. At 24 h, Panobinostat (Cayman Chemical: 13280), a pan HDAC inhibitor, was added (final concentration 25 nM). DMSO was used as a control. Cells were harvested at 48 h and probed for acetylation using Western blotting (antibody: anti-acetylated-lysine (Cell Signaling 9441)).
5
Western Blot Analysis of Protein Fractions
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Protein samples were harvested from H9c2 cardiomyocytes after H/R and the myocardium at 24 hours after resuscitation. The separation of cytosolic and nuclear fractions was performed by centrifugation of samples at 16 000g. Subsequently, the samples were separated by SDS‐PAGE, then transferred to a polyvinylidene fluoride membrane, and finally blocked with 5% nonfat milk. After that, the membranes were incubated with primary anti‐NLRP3 (1:1000, Proteintech, Rosemount, IL), anticleaved caspase‐1 (1:1000, Cell Signaling Technology Inc., Danvers, MA), anti–gasdermin D (1:1000, Proteintech), anti‐HDAC6 (1:1000, Proteintech), anti‐GAPDH (1:5000, BBI Life Science Corporation, Shanghai, China), antiacetylated α‐tubulin (1:1000, Proteintech), anti–α‐tubulin (1:1000, Proteintech), anti‐TFEB (1:1000, Proteintech), antiacetylated lysine (1:1000, Cell Signaling Technology Inc.), antihistone H3 (1:1000, Abcam) at 4 °C for 24 hours, then rinsed with TBST solution, and finally incubated with the secondary antibody (1:5000, BBI Life Science Corporation) at room temperature for 1 hour. The protein was visualized with enhanced chemiluminescence substrates and analyzed by Image J software (National Institutes of Health, Bethesda, MD).
6
Acetylated Proteoform Analysis in Muscular Dystrophy
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Qualitative analysis of acetylated proteoforms was conducted on 2-D DIGE total protein extracts. 2-D immunoblotting was carried out on DMD, BMD1 and BMD2 pooled samples by subjecting each pool (150 μg) to isoelectrofocusing on 18 cm, 6–10 pH-gradient IPG strips (GE Healthcare), with a voltage gradient ranging from 300 to 8000 V, for a total of 52,000 Vh, using an IPGphor electrophoresis unit (GE Healthcare). After focusing, proteins were reduced and alkylated. The second dimension was carried out in 20 × 25 cm2, 12% polyacrylamide gels at 20 °C. Blots were blocked in 5% BSA for 30 min and incubated with a 1:1 mixture of rabbit anti-acetylated-lysine (Ac-K2–100) (1:1000, Cell Signaling Technology, Danvers, MA, USA, #9814) and anti-acetylated-lysine (1:1000, Cell Signaling Technology, #9441) primary antibodies. After washing, membranes were incubated with anti-rabbit HRP-conjugated secondary antibody (1:10,000, GE Healthcare). Signals were visualized by chemiluminescence using the ECL Prime (GE Healthcare) detection kit and the Image Quant LAS 4000 (GE Healthcare) analysis system.
7
Mitochondrial Dynamics Regulation Assay
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LPS was purchased from Sigma (MO, USA). Anti‐Sirt3, anti‐Cyt‐c and anti‐acetylated lysine were obtained from Cell Signalling Technology (MA, USA). Immunofluorescent Anti‐Sirt3 was purchased from Santa Cruz (CA, USA). Anti‐OPA1, anti‐Drp1, anti‐VDAC, anti‐TOM‐20 and anti‐GAPDH were purchased from Abcam (MA, USA). Anti‐Bax and anti‐YME1L1 were purchased from Proteintech (Wuhan, China). Anti‐Flag, Alexa Fluor 488‐ and 594‐conjugated fluorescent secondary antibodies and HRP‐conjugated secondary antibodies were purchased from Antgene (Wuhan, China).
8
Western Blot Analysis of Cell Signaling
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Cells and tissues were lysed by homogenization in RIPA lysis buffer (ThermoFisher Scientific, #89900). Protein extracts were separated by SDS-PAGE and then transferred onto PVDF membrane (Millipore, #IPVH00010). Following overnight incubation with the indicated primary antibodies at 4°C, membranes were incubated with IR dye-coupled secondary antibodies (LI-COR) and then visualized by using the LI-COR Odyssey infrared imaging system (LI-COR). The primary antibodies and diluted ratio include: anti-phospho-AKT 1:1000 (Ser473; Cell Signaling Technology, #9271); anti-AKT 1:1000 (Cell Signaling Technology, #2920); anti-β-ACTIN 1:5000 (Sigma, #A1978); anti-IκBα 1:1000 (Cell signaling Technology, #4814); anti-α-Tubulin 1:1000 (Cell Signaling Technology, #3873); anti-Acetylated Lysine 1:1000 (Cell Signaling Technology, #9441); anti-NFκB p65 1:1000 (Cell Signaling Technology, #8242); anti-p300 1:1000 (Cell Signaling Technology, #86377); anti-FLAG (Sigma, #F1804); anti-Acetyl NFκB p65 (Lys310) 1:1000 (Cell Signaling Technology, #12629). NuRD complex components (CHD4, MTA1, HDAC1, RbAP46) antibodies were all from NuRD Complex Antibody Sampler Kit (Cell Signaling Technology, #8349) and diluted 1:1000 for primary antibody incubation. Detailed information of commercial manufacturers and validation of the antibodies can be found in the Reporting Summary.
9
Acetylation-Dependent Protein Regulation
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All chemicals were obtained from Sigma-Aldrich unless stated otherwise. Enhanced chemiluminesence (ECL) reagents were purchased from NEN, Inc. EDTA-free protease inhibitor tablet was purchased from Roche, Inc. Anti-Acetylated lysine, anti-Ac-p65 and anti-Foxo1 were purchased from Cell Signaling. Anti-Ac-Foxo1/3 was obtained from Santa Cruz Biotechnology.
10
Chidamide Acetylation and Ubiquitination
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Chidamide was synthesized by Shenzhen Chipscreen Biosciences Ltd. (Shenzhen, China) and dissolved in dimethyl sulfoxide (DMSO). MG132 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibody anti-Mcl-1 was purchased from Abcam® (Cambridge, MA, USA), anti-Acetylated-Lysine and anti-β-actin were obtained from Cell Signaling Technology (Boston, MA, USA), and anti-ubiquitin was purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA).
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