Cells on petri dish were washed with PBS then lysed with 1X SDS-PAGE sample loading buffer diluted from 4X loading buffer (250 mM Tris-HCl pH 6.8; 8% SDS; 0.2% Bromophenol Blue; 20% β-mercaptoethanol; 40% glycerol). Whole cell lysates were heated at 95°C for 5 min and separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane. Western blotting was conducted by blocking the membranes in 5% non-fat milk then probing with primary antibodies in 5% non-fat milk for 1 h. Membranes were washed extensively with PBST (phosphate buffered saline supplemented with 0.5% Tween 20), then incubated with HRP-conjugated secondary antibodies for 30 min. After extensive wash with PBST, bound HRP-conjugated secondary antibody was detected by enhanced chemiluminescence (ECL). Antibodies against human mTOR (ab2732), mTOR-S2481 (ab137133), S6K1 (ab9366), S6K-T389 (ab2571), RAGC (ab226199), RAB1A (ab241132), RAB5 (ab18211), and Tubulin (ab15568) were purchased from Abcam. Antibody against LC3 (848801) was from BioLegend.
Ab15568
Manufactured by Abcam
Sourced in United Kingdom
Ab15568 is a monoclonal antibody product manufactured by Abcam. It is designed for use in various laboratory techniques.
Automatically generated - may contain errors
Lab products found in correlation
D1306, by Thermo Fisher Scientific (2 mentions)
Ab14734, by Abcam (2 mentions)
Ab167453, by Abcam (2 mentions)
Ab184224, by Abcam (2 mentions)
Ab133256, by Abcam (2 mentions)
Novex 10 20 tricine protein gels, by Thermo Fisher Scientific (2 mentions)
Ab46798, by Abcam (2 mentions)
A11029, by Thermo Fisher Scientific (2 mentions)
A21429, by Thermo Fisher Scientific (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Ab2732, by Abcam (1 mentions)
Ab137133, by Abcam (1 mentions)
Ab9366, by Abcam (1 mentions)
Ab2571, by Abcam (1 mentions)
Ab18211, by Abcam (1 mentions)
Ab4448, by Abcam (1 mentions)
Ab84870, by Abcam (1 mentions)
A11034, by Thermo Fisher Scientific (1 mentions)
23 protocols using ab15568
1
Comprehensive Western Blot Analysis
2
Immunostaining of Expanded Samples
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Punches were blocked in IF buffer (1% BSA and 0.05% Tween‐20 in PBS) for 1–2 h at RT, followed by the incubation with primary antibody diluted in IF buffer, for 24 h at 4°C. Punches were washed in 1× PBS for 1–2 h and incubated for 24 h at 4°C with secondary antibody and DAPI (ThermoFisher; D1306) diluted 1:10 000 in IF buffer. The following primary antibodies were used for immunostaining of expanded samples: mouse anti‐acetylated tubulin (Sigma, T7451) at 1:4000, mouse anti‐alpha tubulin (Sigma; T6074) at 1:500, rabbit antibeta tubulin (Abcam; ab15568) at 1:250, rabbit anti‐ARL13B (Abcam; ab83879) at 1:150, rabbit anti‐Cep164 (Proteintech; 22227‐1‐AP) at 1:500, rabbit anti‐ANKRD26 (GeneTex; GTX128255) at 1:200, rabbit anti‐FBF1 (sigma; HPA023677) at 1:100, rabbit anti‐RPGRIP1L (Proteintech; 55160‐1‐AP) at 1:150, rabbit anti‐IFT88 (Proteintech; 13967‐1‐AP) at 1:150, mouse anti‐Rootletin (Santa Cruz; sc‐374056) at 1:50, rabbit antipericentrin (Abcam; ab4448) at 1:400, rabbit anti‐Cep290 (Abcam; ab84870) at 1:150 and rabbit antipolyglutamylated tubulin (AdipoGen; AG‐25B‐0030) at 1:800. Secondary antibodies antimouse Alexa Fluor 488 (Invitrogen; A11029), antirabbit Alexa Fluor 488 (Invitrogen; A11034), antimouse Alexa Fluor 555 (Invitrogen; A28180), antirabbit Alexa Fluor 555 (Invitrogen; A21429) were used at a 1:800 dilution to label primary antibodies.
3
Western Blot Analysis of Cell Signaling Proteins
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The SMCs in each group were solicited using the Sonics Vibracell sonicator with a 0.4 mm diameter probe. Proteins in each sample were determined by a Bradford assay (Bio-Rad, Hercules, USA). The samples were denatured at 95°C for 3 min in 1x Nu Page sodium dodecyl sulphate sample buffer (Life Technologies, Carlsbad, USA). Equal amounts of protein were loaded, electrophoresed, and transferred to Immobilon-P membranes (Merch Millipore, Darmstadt, Germany). The membranes were blocked overnight in 5% BSA, followed by 3 h incubation in primary antibodies (0.5 μg/mL) at room temperature. Then the membranes were washed and incubated with secondary antibodies for 1 hour. Antibodies for phospholipase C-beta 1 (ab77743, 1 : 300), protein kinase C (ab23511, 1 : 300), adenylate cyclase (ab124241, 1 : 300), and β-tubulin (ab15568, 1 : 1000) were from Abcam (Cambridge, UK). The protein of β-tubulin was used as internal housekeeping protein. Immunoreactivity was detected using a chemiluminescence's system and quantified by using Image J software (Bio-Rad, Hercules, USA).
4
Western Blot Analysis of Mitochondrial Proteins
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Laemmli buffer containing 4% SDS, 10% β-mercaptoethanol (M6250; Sigma-Aldrich), 20% glycerol (GI345; Melford), 0.004% blue bromophenol (A2331,0025; AppliChem), and 0.125 M Tris-HCl was added to protein lysates, followed by boiling at 95°C for 10 min. Samples were separated on SDS-PAGE and transferred to a nitrocellulose (Macherey-Nagel) or a polyvinylidene difluoride (Merck) membrane. The membranes were blocked with 5% milk (A0830; PanReac AppliChem) and probed with the following antibodies: anti–β-Actin (ab8227; Abcam), anti–β-Tubulin (ab15568; Abcam), anti-CBS (14787-1-AP; Proteintech), anti-CSE (12217-1-AP; Proteintech), anti-MPST (HPA001240; Atlas Antibodies), anti-MTCO1 (ab14705; Abcam), anti-Citrate Synthase (ab96600; Abcam), anti-SOD2 (13141; Cell Signaling), anti-TOM40L (ab236421; Abcam), anti-TIM50 (ab109436; Abcam), anti-HIF1α (sc-13515; Santa Cruz Biotechnology), anti-H3 (ab176842; Abcam), anti-V5 (Merck), anti-HA (H6908; Sigma-Aldrich), and anti-Biotin (HRP conjugate; 5571; Cell Signaling). Immunoblots were next incubated with a secondary antibody (anti-rabbit [AP132P; Merck] or anti-mouse [7076; Cell Signaling]) and visualized using the Western HRP substrate (Merck). ImageJ software was used to quantify the expression levels of the proteins.
5
Immunofluorescence Assay of PRUNE in SH-SY5Y Cells
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SH-SY5Y inducible clones were plated and grown for 24 h in the presence of doxycycline. Then, inducible cells growing on the coverslips were incubated on ice for 1 h and then at 30°C for 2 min. Cells were fixed in 4% paraformaldehyde and permeabilized for 10 min in phosphate buffer containing 0.1% Triton™ X-100, before incubation with 0.1% Triton™ X-100, 10% pig serum and either anti-β-Tubulin (1 µg/ml; Abcam, ab15568), anti-PRUNE (1 µg/ml; Abcam, ab88613) antibodies. Confocal microscopy was carried out using a laser scanning confocal microscope LSM 510 META, Zeiss, with 40×/63× water immersion objectives.
6
Immunoblotting Analysis of Viral Proteins
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Lysates from the above mentioned assays were analyzed by SDS-PAGE, using standard 12% SDS-PAGE to resolve mCherry and its XP-fusion variants, and precast Novex™ 10–20% tricine protein gels (Thermo Fisher) to resolve XPs and enterovirus 2B. Proteins were then transferred to 0.2 µm nitrocellulose membranes and blocked with 4% Marvel milk powder in phosphate-buffered saline (PBS). Immunoblotting of mCherry was performed using anti-mCherry antibody (Abcam, ab167453, 1:3000). A custom rabbit polyclonal antibody raised against XP peptide SNSGNRVSQDQNLQ (GenScript; only able to detect strongly overexpressed XP, 1:250) and an anti-Strep mouse antibody (Abcam, ab184224, 1:1000) were used for detecting HAstV1 XP and Strep-tagged proteins, respectively. The following antibodies were used for cellular targets: anti-tubulin (Abcam, ab15568, 1:500), anti-VDAC1 (Abcam, ab14734, 1:1000), and anti-LAMIN A + C (Abcam, ab133256, 1:3000). Immunoblots were imaged on a LI-COR ODYSSEY CLx imager and analyzed using Image Studio™ version 5.2.
7
SDS-PAGE Protein Separation and Immunoblotting
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Thirty micrograms of protein samples from LSTs and other groups were separated on a 10% or 12% SDS-PAGE and transferred to nitrocellulose membranes (Millipore). Protein bands were visualized using a conventional enhanced chemiluminescence (ECL) system (GE Healthcare, USA) and quantified with Quantity One (Bio-Rad, USA). Primary antibodies used were anti-LCN-2 antibody (ab23477, 1:500; Abcam), anti-MMP-9 (PAB19095, 1:200; Abnova), and anti-beta tubulin antibody (ab15568, 1:200; Abcam).
8
Immunoblot Analysis of Enterovirus Proteins
9
Antibody Immunoprecipitation and Western Blot Analysis
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The following antibodies were used for immunoprecipitation and/or microfluidic western blot analysis by ProteinSimple (PS), immunohistochemistry (IHC), immunofluorescence (IF): CD31 (553371, MEC13.3, BD Biosciences; 1:500 for IF), laminin (ab11575, Abcam; 1:400 for IF), involucrin (ab28057, Abcam; 1:100 for IHC), FAK (06–543, EMD Millipore; 1:50 for PS; 1:100 for IF), paxillin (610051, 349, BD Biosciences, 1:100 for IF), phospho-FAK Y397 (AF4528, R&D Systems; 1:200 for PS), phospho-FAK Y397 (44-624G, Thermo Fisher Scientific; 1:100 for IF), β-tubulin (ab15568; Abcam; 1:50 for PS), SRC (2109, 36D10, Cell Signaling Technology; 1:50 for PS), phospho-SRC Y416 (2101, Cell Signaling Technology; 1:10 for PS).
10
Immunostaining and Expansion Microscopy
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Punches were blocked in IF buffer (1% BSA; [Sigma-Aldrich; A9647] and 0.05% Tween-20 [Sigma-Aldrich; P9416] in 1× PBS) for 1–2 h at RT, and incubated with primary antibody diluted in IF buffer for 24 h at 4°C. Punches were washed in 1× PBS for 1–2 h and incubated with secondary antibody and DAPI (1:10,000 final dilution; Thermo Fisher Scientific; D1306) in IF buffer for 24 h at 4°C. The following primary antibodies were used for immunostaining: mouse anti-acetylated tubulin (Sigma-Aldrich; T7451) at 1:4,000, rabbit anti-β tubulin (Abcam; ab15568) at 1:250, rabbit anti-ARL13B (Abcam; ab83879) at 1:500, rabbit anti-Cep290 (Abcam; ab84870) at 1:600, and rabbit anti-Cep164 (Proteintech; 22227–1-AP) at 1:500. Secondary antibodies Alexa Fluor 488 anti-mouse (Thermo Fisher Scientific; A11029) and Alexa Fluor 555 anti-rabbit (Thermo Fisher Scientific; A21429) were used at a 1:800 dilution to label primary antibodies for 24 h. After immunostaining, the samples were expanded in deionized H2O for 2 h at RT with deionized H2O exchanged every 10 min and additionally overnight at 4°C. Prior to imaging, expanded punches were mounted in Rose chambers, Attofluor cell chambers, or glass-bottom Microwell dishes (MatTek; P35G-1.5-14-C).
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