To measure protein levels, cell lysates were resolved by denaturing gel electrophoresis before electrotransfering to Protran nitrocellulose membrane (Millipore, USA). The membrane was subjected to Western blot analysis with antibodies against proteins of interest. The signals from the primary antibody were amplified by horse radish peroxidase (HRP)-conjugated anti-mouse IgG (GE, USA) or anti-rabbit IgG (GE, USA), and detected with Enhanced Chemiluminescence Plus (GE, USA). The following antibodies and dilutions were used: rabbit anti-Foxm1 (1∶500; Santa cruz SC-502), rabbit anti-Nanog (1∶1000; Millipore AB9220), rabbit anti-Sox2 (1∶1000; Abcam AB59776), mouse anti-Oct4 (1∶1000; Cell signaling #4286), rabbit anti-Stat3 (1∶500; Santa cruz SC-482), rabbit anti-p-Stat3 (1∶500; Santa cruz SC-8059), rabbit anti-Klf4 (1∶500; Santa cruz SC-20691), mouse anti-β-actin (1∶5000; Beyotime AA128), mouse anti-GFP (1∶1000; Beyotime AG279).
Protran nitrocellulose membrane
Manufactured by Merck Group
Sourced in United States
Protran nitrocellulose membranes are a type of laboratory equipment used for protein and nucleic acid detection and analysis. They provide a stable, high-binding surface for the immobilization of biomolecules. The membranes are made of nitrocellulose, a material known for its strong binding properties.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ecl kit, by Thermo Fisher Scientific (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Adi spa 827, by Enzo Life Sciences (2 mentions)
Protease inhibitors cocktail, by Roche (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Ab9220, by Abcam (1 mentions)
Enhanced chemiluminescence plus, by GE Healthcare (1 mentions)
Hrp conjugated anti mouse igg, by GE Healthcare (1 mentions)
Mouse anti gfp, by Beyotime (1 mentions)
Rabbit anti stat3, by Santa Cruz Biotechnology (1 mentions)
Mouse anti β actin, by Beyotime (1 mentions)
Rabbit anti foxm1, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit igg, by GE Healthcare (1 mentions)
Ab19194, by Abcam (1 mentions)
Ab25375, by Abcam (1 mentions)
Ab18981, by Abcam (1 mentions)
Ab14935, by Abcam (1 mentions)
Ab178424, by Abcam (1 mentions)
Ab134047, by Abcam (1 mentions)
Enhanced chemiluminescence reagent, by PerkinElmer (1 mentions)
17 protocols using protran nitrocellulose membrane
1
Protein Expression Analysis by Western Blot
2
Western Blot Analysis of Neuroinflammatory Markers
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After i.c.v. LPS injection, the mice were anesthetized and perfused with ice-cold PBS to clear blood-borne proteins. Next, the brain was homogenized in 1 ml of cold PBS on ice, and the homogenate was centrifuged (12,000 rpm, 5 min). Cells were digested with radioimmunoprecipitation assay (RIPA) lysis buffer (50 mmol/L Tris–HCl, 150 mmol/L NaCl, 1 % Nonidet-40, 0.5 % sodium deoxycholate, 1 mmol/L EDTA, 1 mmol/L PMSF) for 30 min on ice and centrifuged at 12,000 rpm for 15 min at 4 °C. The brain homogenates or cell lysate were diluted in PBS and loading buffer, boiled (100 °C, 10 min), loaded on a 10 % acrylamide–SDS gel, and transferred to a Protran nitrocellulose membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5 % dry milk in PBS for 2 h at room temperature, incubated in primary antibodies against P-selectin (ab178424, Abcam, Cambridge, USA), E-selectin (ab18981, Abcam),VCAM-1 (ab134047, Abcam), ICAM-1 (ab25375, Abcam), CXCR2 (ab14935, Abcam), albumin (ab19194, Abcam), and β-actin (Cell Signaling, Beverly, CA, USA) overnight at 4 °C, washed, incubated in species-appropriate HRP-conjugated secondary antibodies for 1–2 h at room temperature in the dark, and washed three times. Then, the membranes were subjected to immunodetection using enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA, USA).
3
Phosphorylation-Specific Antibody Validation
4
Western Blot Analysis of Fluorescent Proteins
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NaOH/TCA protein extracts [99 (link)] were prepared and subjected to SDS-PAGE. Resolved proteins were transferred to PROTRAN nitrocellulose membrane (Sigma-Aldrich/Merck, Darmstadt, Germany) or PVDF (Sigma-Aldrich/Merck, Darmstadt, Germany). Membranes were blocked with 5% non-fat milk (Regilait, Saint-Martin-Belle-Roche, France) and probed with corresponding antibodies. Red fluorescent protein fusions were detected with an anti-tRFP antibody (AB233, Evrogen, Moscow, Russia) that recognizes TagRFP-T and mTagBFP, and with an anti-RFP antibody (3F5, Chromotek, Planegg-Martinsried, Germany) recognizing mRFP and mCherry fluorescent proteins. The antibodies were used at dilution 1:1000 at 4 °C overnight. As a secondary antibody, the goat anti-rabbit IgG antibody conjugated to HRP (32460, Pierce-ThermoFisher Scientific, Waltham, MA, USA) and the goat anti-mouse IgG antibody conjugated to HRP (32430, Pierce-ThermoFisher Scientific, Waltham, MA, USA) were used both at dilution 1:600. GFP protein fusions were assayed with the GFP(B-2) HRP conjugate (sc-9996, Santa Cruz Biotechnology, Dallas, TX, USA) at dilution 1:1000, 1 h at RT or overnight at 4 °C. Proteins were detected by SuperSignal West Dura Substrate (Pierce-ThermoFisher Scientific, Waltham, MA, USA).
5
Western Blot Analysis of VEGFR-2 Signaling
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Equivalent protein concentrations were loaded onto 8% polyacrylamide gels and subjected to SDS-PAGE. Proteins were transferred to a Protran nitrocellulose membrane (GE10600003, Sigma) before being incubated in 5% milk powder. Membranes were then incubated overnight in primary antibody diluted 1:1,000 at 4°C. Membranes were washed with 0.1% Tween-20 in PBS and incubated in an appropriate horseradish peroxidase–conjugated secondary antibody (Dako) diluted 1:2,000 for 2 hours at room temperature. Bands were visualized by incubation with a 1:1 solution of Pierce ECL Western Blotting Substrate (34579, Thermo Fisher Scientific). Chemiluminescence was detected on a ChemiDoc MP Imaging System (Bio-Rad). Densitometric readings of band intensities were obtained using ImageJ. Primary antibodies: p-VEGFR-2Y1175 (2478, CST; RRID:AB_331377), VEGFR-2 (2479, CST; RRID:AB_2212507), NRP1 (3725, CST; RRID:AB_2155231), NRP2 (3326, CST), HSC70 (Sc-7298, SCB; RRID:AB_627761), EDA-FN (F6140, Sigma; RRID:AB_476981), Ubiquitin (Sc-8017, SCB; RRID:AB_628423).
6
Protein Extraction and Analysis Protocol
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MAECs were solubilized in lysis buffer (50 mmol/L HEPES pH 7.5, 150 mmol/L NaCl, 10 mmol/L EDTA, 10 mmol/L Na2P2O7, 2 mmol/L Na3VO4, 100mmol/L NaF, 10% glycerol, 1% Triton X-100) for 2 h at 4 °C. Protein lysates were clarified by centrifugation at 16,000× g for 20 min. Cell lysates were then separated by SDS-PAGE and transferred into 0.45 μm Protran Nitrocellulose Membrane (Sigma-Aldrich). Upon incubation with primary and secondary antibodies (for a full list of the antibodies, see above), immunoreactive bands were detected by chemiluminescence and densitometric analysis was performed using ImageJ software.
7
Cell Lysis and Protein Analysis Protocol
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Cells were lysed in cold buffer containing 150 mM NaCl, 20 mM HEPES pH 7.5, 10 mM EDTA and 1% Triton X100, and supplemented with a protease inhibitor cocktail (Roche), Phos Stop Easypack (Roche) and 20 mM NEM . Protein concentration was determined with a standard BCA assay (Pierce). Samples with the same protein concentration were mixed with non‐reducing Laemmli buffer (62.5 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol and 0.01% bromophenol blue) and heated for 5 min at 95°C. For reducing SDS‐PAGE, samples were supplemented with 100 mM DTT. Protein samples separated by either reducing or non‐reducing SDS‐PAGE were then transferred to Protran nitrocellulose membrane (Merck) and probed with the following antibodies: monoclonal mouse anti‐Actin (MAB1501, Sigma Aldrich), monoclonal mouse anti‐FLAG M2 antibody (F3165, Sigma‐Aldrich), monoclonal mouse anti‐KDEL (for BIP detection) (ADI‐SPA‐827, Enzo life Sciences) and polyclonal rabbit anti‐ERO1 alpha (Zito, Chin, et al., 2010 (link)), polyclonal p‐eIF2 alpha (44‐728G, Invitrogen) and eIF2 alpha (AH01182, Invitrogen). The western blots were quantified by acquiring the signal with ChemiDoc MP Imaging System and processing with Image Lab analysis software (Bio‐Rad Laboratories). The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology.
8
Immunoblot Analysis of Cellular Proteins
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Cells were lysed in cold buffer containing 150 mM NaCl, 20 mM HEPES pH 7.5, 10 mM EDTA and 1% Triton X100, supplemented with a protease inhibitors cocktail (Roche) and 20 mM NEM. Protein samples separated by either reducing or non-reducing SDS-PAGE were then transferred to Protran nitrocellulose membrane (Merck) and probed with the following antibodies: monoclonal mouse anti-Actin (MAB1501, Sigma Aldrich), monoclonal mouse anti-KDEL (ADI-SPA-827, Enzo life Sciences), monoclonal mouse anti-FLAG M2 (F3165, Sigma Aldrich), and polyclonal rabbit anti-ERO1 alpha [40 (link)].
9
Cellular Lysis and Protein Analysis
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Cells were lysed in cold buffer containing 150 mM NaCl, 20 mM HEPES pH 7.5, 10 mM EDTA and 1% Triton X-100, and supplemented with protease inhibitors cocktail (Roche) and 20 mM NEM. Protein concentration was determined by standard BCA assay (Pierce). Samples with equal protein concentration were mixed with non-reducing Laemmli buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol and 0.01% bromophenol blue) and heated for 5 min at 95°C. For reducing SDS-PAGE, samples were supplemented with 100 mM DTT. Protein samples separated by either reducing or non-reducing SDS-PAGE were then transferred to Protran nitrocellulose membrane (Merck) and probed with the following antibodies: mouse pan-actin (clone C4) and mouse GAPDH (AB9484, Abcam) from Sigma Aldrich, monoclonal mouse anti-FLAG M2 (F3165, Sigma Aldrich), monoclonal mouse anti-KDEL (ADI-SPA-827, Enzo life Sciences), Selenoprotein N (A-11) from Santa Cruz Biotechnology, mouse OXPHOS (ab110413) from Abcam and rabbit ERO1.49 (link)
10
Protein Expression Analysis by Western Blot
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Proteins were isolated from cell cultures by standard methods. A total amount of 30 μg of protein was loaded on a 6% or 12% SDS-PAGE gel. After electrophoresis, proteins were transferred to protran nitrocellulose membranes (Sigma-Aldrich, Madrid, Spain), and blots were probed with the indicated primary antibodies, as previously described [14 (link)]. The antibodies used were the following: rabbit polyclonal anti-C3 (Abcam, Cambridge, UK), mouse monoclonal anti-C/EBPβ (Abcam, Cambridge, UK), goat polyclonal anti-COX2 (Santa Cruz Biotechnologies, Mountain View, CA, USA), rabbit polyclonal anti-IL1β (Abcam, Cambridge, UK), rabbit polyclonal anti-α-tubulin (Sigma-Aldrich, Madrid, Spain) and secondary peroxidase conjugated antibodies (Jackson Immunoresearch, West Grove, PA, USA). Quantification was performed using Scion Image software (Scion Corporation, http://scion-image.software.informer.com/ ).
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