The effect of PMF on protein expression was evaluated using Western blot. MCF-7 cells were seeded in 6-cm petri dishes until 80% confluency, and then, cells were treated with the test compound (0–20 μM) for 5 h. Cells were lysed using PhosphoSafe extraction reagent obtained from Novagen (Madison, WI, USA). After protein quantification using a BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA), 20 μg of protein of each sample were separated by electrophoresis using Nu-PAGE 10% SDS-PAGE Bis-Tris gel. Then, proteins were transferred to a polyvinylidene fluoride membrane and blocked (1 h) with 3% bovine serum albumin. Membranes were incubated overnight with primary antibodies (1:1000) of BCL-2 (15071), cytochrome c (Cyt c, 11940), BAX (2772), caspase-3 (9662), PARP-1 (5625), p21WAF1/CIP1 (p21, 12D1), p53 (9282), NF-κB p65 (8242, Cell Signaling Technology, Inc., Danvers, MA, USA) and β-actin (sc-130656, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The membranes were also incubated for 1 h with an anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology, Inc.) and detection of proteins was achieved via a chemiluminescence horseradish peroxidase (HRP)-conjugated (Santa Cruz Biotechnology, Inc.) reagent. The band densities were analyzed using Image J (Bethesda, MD, USA).
Anti rabbit or anti mouse secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
Anti-rabbit or anti-mouse secondary antibodies are laboratory reagents used to detect the presence of primary antibodies that have been raised against rabbit or mouse antigens. These secondary antibodies are conjugated with enzymes, fluorescent dyes, or other reporter molecules, allowing for the visualization and amplification of the signal generated by the primary antibody-antigen interaction. They are commonly used in a variety of immunoassay techniques, such as Western blotting, ELISA, and immunohistochemistry.
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Lab products found in correlation
β actin, by Santa Cruz Biotechnology (4 mentions)
Ecl advance, by GE Healthcare (4 mentions)
Polyvinylidene difluoride membrane, by Cytiva (3 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (2 mentions)
β actin, by Merck Group (2 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti synaptophysin, by Merck Group (1 mentions)
Anti α tubulin, by Abcam (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Image acquisition and analysis software, by Bio-Rad (1 mentions)
Nf κb, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Wnt5a, by Proteintech (1 mentions)
Sodium dodecyl sulfate loading buffer, by Beyotime (1 mentions)
Proliferating cell nuclear antigen (pcna), by Proteintech (1 mentions)
Antibody diluent, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
21 protocols using anti rabbit or anti mouse secondary antibody
1
Evaluating PMF Effects on Protein Expression
2
Synapse Protein Analysis in Brain Tissue
3
Protein Expression Analysis in Cardiac Tissues
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Mouse left ventricle tissues, H9C2 cells, and ECs were collected and sonicated in lysis buffer. The homogenates were centrifuged at 16 000g at 4°C for 15 minutes. The protein concentrations were analyzed with a BCA protein assay kit (Pierce Co, Rockford, IL). An aliquot (30 µg for heart tissues and 25 µg for cells) of protein samples was separated by 10% SDS‐PAGE gel and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% dry milk in Tris‐buffered saline and incubated with the following primary antibodies overnight: p300 (1:1000; Cell Signaling), acetyl‐histone H3 (lys56) (H3K56ac, 1:1000; Cell Signaling), total lysine acetylation (1:1000; Cell Signaling), β‐MHC (β‐myosin heavy chain; 1:1000; Abcam, Cambridge, MA), arginase I and II (1:1000; Novus Bio, Littleton, CO), NF‐κB, TLR‐4 (toll‐like receptor 4), MyD88 (myeloid differentiation primary response 88; 1:1000; Santa Cruz, CA), eNOS (1:1000; BD Transduction, San Jose, CA), VCAM‐1 and IRAK‐4 (interleukin‐1 receptor‐associated kinase 4; 1:1000; Cell Signaling). After washing, the membranes were incubated for 2 hours with an anti‐rabbit or anti‐mouse secondary antibody coupled to horseradish peroxidase (1:5000; Santa Cruz). Densitometric analyses were performed with image acquisition and analysis software (Bio‐Rad, Hercules, CA).
4
Protein Extraction and Western Blot Analysis
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Whole cell protein extracts were prepared by lysis of cells using cold RIPA buffer (100 µl/well, Beyotime, Shanghai, China). The concentration was measured using a bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). Equal protein amounts of each of the samples were denatured with 6 × sodium dodecyl sulfate (SDS) loading buffer (Beyotime, Shanghai, China) at 100 C for 10 min. Protein samples were separated by electrophoresis on a 10% polyacrylamide SDS gel (Beyotime, Shanghai, China) and transferred onto 0.45-µm nitrocellulose membranes (Beyotime, Shanghai, China). Following 60 min of blocking with 5% fat-free milk in double distillation H 2 O, membranes were incubated with the primary antibodies in antibody diluent (Beyotime, Shanghai, China) overnight at 4 °C. Blots were washed with PBST (PBS with Tween), and incubated for 1 hr with the anti-rabbit or anti-mouse secondary antibody, as appropriate (1:2000, Santa Cruz Biotechnology, USA). Immunoreactive protein bands were detected with an Odyssey Scanning system (Li-Cor, USA). The density of the bands was measured by ImageStudio.
The following primary antibodies and dilutions were used:
β-actin (1:2000, Santa Cruz Biotechnology, USA), LATS2 (1:1000, Bioworld, Shanghai, China), PCNA (1:2000, Danvers, MA, USA) and WNT5A (1:1000, Proteintech, Chicago, USA).
The following primary antibodies and dilutions were used:
β-actin (1:2000, Santa Cruz Biotechnology, USA), LATS2 (1:1000, Bioworld, Shanghai, China), PCNA (1:2000, Danvers, MA, USA) and WNT5A (1:1000, Proteintech, Chicago, USA).
5
Western Blotting of 3T3-L1 Cell Lysates
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3T3-L1 cells were harvested and lysed in ice-cold lysis buffer containing a protease inhibitor cocktail and 1 mM phenylmethanesulfonyl fluoride for 30 min, followed by centrifugation at 10,000 × g for 30 min at 4 °C. Proteins (50 μg) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Amersham, UK). The membranes were incubated with primary antibodies, followed by incubation with anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology) and protein bands were visualized using an enhanced chemiluminescence system (ECL Advance, GE Healthcare, Hatfield, UK).
6
Western Blot Analysis of Protein Lysates
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The protein lysates were prepared from mice liver or HepG2 and AML-12 cells using Pro-Prep Protein Extraction Solution (Intron Biotechnology, Seoul, Korea), according to the manufacturer’s instructions. Equal amounts of protein (50 μg) were separated by 12% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Amersham, UK). After transfer, the membranes were blocked in 5% nonfat skim milk, followed by incubation with primary antibodies (1:1000) overnight at 4 °C. After over night incubation, membranes were probed with anti-rabbit or anti-mouse secondary antibodies (1:1000) (Santa Cruz Biotechnology) conjugated to peroxidase, and the protein bands were detected by using an enhanced chemiluminescence system (ECL Advance, GE Healthcare, Hatfield, UK).
7
Neuroprotective Mechanism of TC-G 1008
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The expression levels of GPR39, SIRT1, PGC-1α and Nrf2 were measured at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h and 7 days post-HIE by Western blot following the manufacturer’s recommendations [34 (link), 35 (link)]. To analyze whether GPR39 receptor and SIRT1/PGC-1α/Nrf2 pathway were involved in the neuroprotective effects of TC-G 1008, the expression levels of GPR39, SIRT1, PGC-1α and Nrf2, and pivotal inflammatory cytokines IL-6, IL-1β, TNF-α were assessed via Western blot. RIPA lysis buffer (Santa Cruz Biotechnology, USA) was used to obtain whole cell lysates. Primary antibodies used were rabbit anti-GPR39 (1:500, Bioss), mouse anti-SIRT1 (1:2000, Abcam), rabbit anti-PGC-1α (1:1000, Abcam), rabbit anti-Nrf2 (1:1000, Abcam), rabbit anti-interleukin (IL)-1β (1:1000, Abcam), rabbit anti-interleukin (IL)-6 (1:1000, Abcam), mouse anti-TNF-α(1:500, Abcam) and mouse anti-β-actin(1:3000, Santa Cruz). The next day, the anti-rabbit (or anti-mouse) secondary antibodies (1:3000, Santa Cruz Biotechnology, USA) were incubated at room temperature for 1–2 h. The gray values were quantified and analyzed by Image J software (NIH).
8
Protein Expression Analysis by Western Blot
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After treatment with different concentrations of CuE (0, 1 and 2.5 μM) for 24 h, cells were washed twice with cold PBS and harvested using cell scrapes and quantified by BCA method. Equal amounts of protein extracts were loaded on to SDS/PAGE and ran at 100 mV for 80 min, followed by transferring to PVDF membranes at 100 mV for 30 min at room temperature. The membrane was blocked in 5% non-fat milk, and then incubated with primary antibodies as indicated. Subsequent to being incubated with anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology) for 1 h, the immune complexes were detected using the enhanced chemiluminescence (ECL) method. Blot intensity was quantified though the Image J software.
9
Western Blot Analysis of Receptor Proteins
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Protein density by Western blot analysis of the P2X7R, A2AR, and A1R receptors was performed based on a prior work [36 (link)]. In brief, cells were initially homogenized in ice-cold radioimmunoprecipitation assay (RIPA) buffer added with 1 mM phosphatase and protease inhibitors and centrifuged at 12,000 rpm at 4°C for 10 min. Subsequently, samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immun-Blot® PVDF membranes (Bio-Rad Laboratories). After blocking, membranes were incubated overnight at 4°C with the primary antibodies: P2X7R (dilution 1 : 500), A2AR (dilution 1 : 800), and A1R (dilution 1 : 500), all obtained from Santa Cruz Biotechnology, Inc. After this step, membranes were washed with Tris-buffered saline (pH 7.6) with 0.1% Tween 20 (TBST) and further incubated with anti-rabbit or anti-mouse secondary antibodies (dilution 1 : 10.000, Santa Cruz Biotechnology, Inc.) at room temperature for 90 min. The membranes were washed again, incubated with an enhanced chemifluorescent substrate (Immobilon® Forte Western HRP Substrate, Merck KGaA), and analyzed with a ChemiDoc Imaging System (Bio-Rad Laboratories). As a control for protein concentration, membranes were reprobed and tested for β-actin immunoreactivity (dilution 1 : 1000, Santa Cruz Biotechnology, Inc.).
10
Western Blot Protein Analysis
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Proteins (40 μg per well) were separated from HepG2 cells using SDS-PAGE on 8% gels and transferred to polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies, followed by incubation with anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) and protein bands were visualized using an enhanced chemiluminescence system (ECL Advance, GE Healthcare, Hatfield, UK).
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