Protein lysates from cells were obtained as previously described [15 (link)]. Cells were washed once in ice-cold Tris-buffered saline (TBS), lysed for 30 minutes in TKM-buffer (50 mM Tris–HCl pH 7.5, 1% NP40, 25 mM KCl, 5 mM MgCl2, 1 mM NaVO4, 5 mM NaF, 20 μg/ml each Leupeptin/Aprotinin) and nuclei were removed by centrifugation. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to standard procedures. Proteins were blotted onto polyvinylidene difluoride (PVDF, Immobilon-P, Merck-Millipore, Darmstadt, Germany) membranes. Membranes were blocked with 10% BSA in PBS. For protein detection the membranes were incubated with primary antibodies for L-plastin (clone LPL4A.1, Neomarkers) (1 μg/ml), phospho L-plastin (our own antibody) or ß-actin (clone A2066, Sigma-Aldrich) (1:900) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Jackson ImmunoResearch). Proteins were visualized with ECL (Amersham-Pharmacia). Densitometric analysis was performed using a GS-800-densitometer and “Quantitiy One” software (BioRad). L-plastin phosphorylation was calculated as the ratio between phospho-protein and total protein. Data from densitometric analysis are reported as mean ± SEM.
Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibodies
Manufactured by Jackson ImmunoResearch
Sourced in United States
Horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies are laboratory reagents used to detect and quantify target proteins in various analytical techniques, such as Western blotting, ELISA, and immunohistochemistry. These antibodies are conjugated with the enzyme horseradish peroxidase, which enables the visualization and amplification of the target protein signal.
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Lab products found in correlation
Gs 800 densitometer, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Fusion solo system, by Vilber (1 mentions)
Rabbit anti erbb2, by Cell Signaling Technology (1 mentions)
Fusion software, by Vilber (1 mentions)
Anti smc3, by Fortis Life Sciences (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Antibody cross linking kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor mixture, by Thermo Fisher Scientific (1 mentions)
Gradient sds page gel, by Bio-Rad (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Anti β tubulin, by Abcam (1 mentions)
Anti p p38, by Cell Signaling Technology (1 mentions)
Anti pmca4 ja9, by Merck Group (1 mentions)
Anti phospho p44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions)
9 protocols using horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibodies
1
SDS-PAGE Immunoblotting of L-plastin
2
Quantitative Western Blot Analysis of Phosphoproteins
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The cell lysates were separated by electrophoresis in 8% SDS-PAGE gel followed by blotting onto ECL-grade nitrocellulose (Amersham, GE Healthcare, Chicago, IL, USA) as described in [26 (link)]. The blots were probed with monoclonal anti-phosphotyrosine antibody 4G10 (Millipore, Merck KGaA, Darmstadt, Germany) and with rabbit anti-phosphoErbB2 (Cell Signaling Technology, Leiden, Netherlands) and rabbit anti-ErbB2 (Cell Signaling Technology) antibodies. Blots were blocked overnight in 5% non-fat milk or 1% BSA (for phosphoprotein detection) in a TBST buffer (10 mM Tris-HCl, pH 7.8, 150 mM NaCl, and 0.1% Tween 20) and then incubated with primary antibodies. After incubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA), immunoreactive bands were visualized by enhanced chemiluminescence (Pierce, New Brighton, MN, USA). For the quantitative analysis of Western blots, we used the Fusion Solo system (Vilber Lourmat, France). The captured images were manually selected in rectangles and further analyzed by densitometry with Fusion software (Vilber Lourmat), the background was subtracted by selecting non-stained blot areas. Final calculations were made using GraphPad 6.0.1 software (GraphPad Software, La Jolla, CA, USA).
3
Immunoprecipitation and Western Blot Analysis
4
Western Blot Analysis of Liver Proteins
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Liver samples were lysed with ice-cold RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 10 mM β-glycerophosphate, 5 mM sodium pyrophosphate dibasic and 1% Nonidet P-40) in the presence of protease and phosphatase inhibitor mixtures (Thermo Fisher Scientific), 1 mM DTT and 2 mM Na3VO4. Total sample lysates were mixed with 6× SDS loading buffer (Alfa-Aesar) and boiled for 5 min. Protein samples (10–100 μg) were loaded and separated on 4–20% gradient SDS/PAGE gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h with 5% BSA in 1× TBS supplemented with 0.1% Tween 20 (Bio-Rad) and incubated with the following antibodies: GS (610518, BD), GLS2 (150474, Abcamab), and GAPDH (14C10, Cell Signaling). Bound antibodies were detected using horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (1:10,000; Jackson ImmunoResearch) and enhanced chemiluminescence reagent (Thermo Fisher Scientific). Band intensities were quantified in ImageJ software.
5
Protein Expression Analysis by Western Blot
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Total proteins were extracted from cells by precipitation with 6% TCA. Samples were resolved by electrophoresis in 10% acrylamide gel and then electroblotted onto PVDF membranes (BioRad, Hercules, CA, USA) as described previously [22 (link)].
Blots were incubated with the following primary antibodies; Mouse monoclonal antibodies: anti-PMCA4b (JA3, 1:1000, cat. # MABN1801, Sigma-Aldrich), anti-PMCA4 (JA9, 1:1000, cat. # P1494, Sigma-Aldrich), anti-HSP (1:1000, cat. # 2402, Cell Signaling Technology, Danvers, MA, USA), anti-PP38 (1:1000, cat. # 9216, Cell Signaling Technology). Rabbit monoclonal anitbodies (Cell Signaling Technology): anti-phospho-p44/ 42 MAPK (ERK1/2) (1:2000, cat. # 4370), anti-ERK1/2 (MK1) (1:1000, cat. # 9102), anti-PHSP27 (1:1000, cat. # 9709), anti-P38 (1:1000, cat. # 9212), anti-β4-integrin (D8P6C)XP (1:1000, cat. # 14803). Rabbit polyclonal antibodies: anti-β-tubulin (1:1000, Abcam, cat. # ab6046). Detection was achieved using horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (dilution 1:10,000, Jackson ImmunoResearch, West Grove, PA, USA) and visualized with Pierce ECL Western Blotting Substrate (Thermo Scientific). In order to measure protein expression levels, densitometry analysis of specific bands was performed using the ImageJ software v1.42q (National Institutes of Health, Bethesda, MD, USA).
Blots were incubated with the following primary antibodies; Mouse monoclonal antibodies: anti-PMCA4b (JA3, 1:1000, cat. # MABN1801, Sigma-Aldrich), anti-PMCA4 (JA9, 1:1000, cat. # P1494, Sigma-Aldrich), anti-HSP (1:1000, cat. # 2402, Cell Signaling Technology, Danvers, MA, USA), anti-PP38 (1:1000, cat. # 9216, Cell Signaling Technology). Rabbit monoclonal anitbodies (Cell Signaling Technology): anti-phospho-p44/ 42 MAPK (ERK1/2) (1:2000, cat. # 4370), anti-ERK1/2 (MK1) (1:1000, cat. # 9102), anti-PHSP27 (1:1000, cat. # 9709), anti-P38 (1:1000, cat. # 9212), anti-β4-integrin (D8P6C)XP (1:1000, cat. # 14803). Rabbit polyclonal antibodies: anti-β-tubulin (1:1000, Abcam, cat. # ab6046). Detection was achieved using horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (dilution 1:10,000, Jackson ImmunoResearch, West Grove, PA, USA) and visualized with Pierce ECL Western Blotting Substrate (Thermo Scientific). In order to measure protein expression levels, densitometry analysis of specific bands was performed using the ImageJ software v1.42q (National Institutes of Health, Bethesda, MD, USA).
6
Western Blot Analysis of Protein Samples
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Harvested cells were lysed using lysis buffer (pH 8.0, 20 mM Tris-HCl, 10% glycerol, 137 mM NaCl, 10 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, phosphatase inhibitor, and protease inhibitor cocktail). Samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Cytiva). The membranes were blocked with 5% skim milk in phosphate-buffered saline-Tween-20 (PBS-T; 140 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 2 mM KH2PO4, and 0.05% Tween-20). Proteins were immunoblotted with the appropriate primary antibody and then horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA). Immunoreactive proteins were detected with ECL solution (ATTO, Tokyo, Japan).
7
Antibody Selection and Characterization
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Antibodies and their manufacturers were as follows: anti-β-actin (AP0060; Bioworld Technology, St. Louis Park, MN, USA), anti-α glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5000) (AT0002; CMCTAG, Dover, DE, USA), anti-GFP (1:5000) (AT0028; CMCTAG), anti-FLAG (1:5000) (AT0022; CMCTAG), anti-LC3 (1:3000) (L7543; Sigma-Aldrich), anti-p62 (1:5000) (P0067; Sigma-Aldrich), and anti-14-3-3ε (1:800) (sc-23957; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Anti-NSP2 antibodies were prepared by immunizing New Zealand white rabbits with a recombinant protein composed of the N-terminal 180 amino acids of NSP2 (NSP2-180). The monoclonal antibody against PRRSV-2 nucleocapsid (N) protein 6D10 was prepared in our laboratory [44 (link)]. Horseradish-peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies were purchased from Jackson Laboratories (1:5000) (West Grove, PA, USA).
Complete™ protease inhibitor cocktail (04693132001) was purchased from Roche (Basel, Switzerland), the aggresome detection kit (ab139486) was purchased from Abcam (Cambridge, UK), and nocodazole (M1404) and MG132 (C2211) were obtained from Sigma-Aldrich.
Complete™ protease inhibitor cocktail (04693132001) was purchased from Roche (Basel, Switzerland), the aggresome detection kit (ab139486) was purchased from Abcam (Cambridge, UK), and nocodazole (M1404) and MG132 (C2211) were obtained from Sigma-Aldrich.
8
Puromycin Incorporation and Translation Regulation
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Cells were treated with 10 µg/mL puromycin (Invitrogen) in growth medium for 5 min, allowing puromycin to incorporate into the de novo synthesized proteome. Cells were then trypsinized to harvest and cell pellets were split to be lysed in TRIzol (Invitrogen) for RNA purification and in NuPAGE LDS sample buffer for Northern blot and Western blot analyses, respectively. For Western blotting, proteins were separated on 10% BisTris PAGE (NuPAGE), and transferred to PVDF membranes (Life Technologies). The membrane was stained with Ponceau to normalize for sample loading, then washed and blocked with 5% nonfat dry milk in TBST. The membranes were probed with 1:1000 of the following primary antibodies: (i) mouse anti-human puromycin antibody (EMD Millipore) to detect de novo protein synthesis, (ii) mouse anti-human total eIF2α (Cell Signaling Technology), and (iii) rabbit anti-human eIF2α-phosphoS51 (Abcam) followed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:10,000, Jackson ImmunoResearch).
9
Western Blot Analysis of CXCL12/CXCR4 Signaling
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Animals were sacrificed by decapitation and the L4/5 spinal dorsal horns were harvested and temporarily stored. Then, the samples were homogenized in ice‐cold RIPA lysis buffer. After measurement of concentration, the protein samples were separated on 10% SDS‐PAGE and electro‐transferred onto PVDF membranes. Protein samples were then incubated with antibodies for rabbit anti‐CXCL12 (1:1000; Abcam), rabbit anti‐CXCR4 (1:1000, Abcam), and mouse anti‐Actin (1:2000; Santa). The membranes were incubated with horseradish peroxidase–conjugated anti‐mouse or anti‐rabbit secondary antibodies (1:2000, Jackson), and exposed to film. The intensity of the selected bands was analyzed using Image J software.
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