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50 protocols using Anti-β-actin

Western blot experiments were performed as previously [67 (link)]. Anti-SRGN (1:100, sc-374657, Santa Cruz Biotechnology), Anti-HIST1H1C (1:1000, ab4086, abcam), Anti-APOE (1:1000, #13366, Cell Signaling Technology), anti-Tubulin (1:5000, #T0023, Affinity Biosciences) and anti-β-Actin (1:4000, #T0022, Affinity Biosciences) were used as primary antibodies. The blots were imaged with the ChemiScope System (Clinx, China).
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Protein concentrations were determined with a BCA protein assay kit (Beyotime, P0012i). Twenty to thirty micrograms of total protein was separated by 10% SDS-PAGE (EpiZyme, pg112) and then transferred onto nitrocellulose membranes (Pall, 66485). Nonspecific sites were blocked with 5% nonfat milk in phosphate-buffered saline (with 0.1% Tween-20) at room temperature. Next, the blots were incubated overnight at 4 ℃ with the following primary antibodies: anti-Hbα (Santa Cruz, sc-514378), anti-Hbβ (Santa Cruz, sc-21757), anti-CD31 (Affinity, AF6191), anti-glutathione peroxidase 4 (GPX4) (Affinity, DF6701), anti-heme oxygenase-1 (HO-1) (Affinity, AF5393), anti-Bcl2 (ImmunoWay, YM3041), anti-Bax (ImmunoWay, YT0455), anti-Notch1 (Cell Signaling Technology, #3608), anti-Jag1 (Cell Signaling Technology, #70109), anti-Hes1 (Abcam, ab108937), anti-Hey1 (Abcam, ab154077), and anti-β-actin (Affinity, AF7018). The next day, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Affinity, s0001) and visualized with New Super ECL assay kit (KeyGen BioTECH, KGP1128).
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3

Western Blot Analysis of Apoptosis Markers in HT22 Cells

To detect the expression levels of apoptosis-related proteins of HT22 cells, Western blot was performed. HT22 cells were collected and total cell protein was extracted from each group. After being quantified and denatured, electrophoresis was performed in a 12% polyacrylamide gel. 20 μg total protein per sample of each group was added, after electrophoresis, transferring to the membrane. The membranes were blocked with 5% skim milk for 2 h and incubated with primary antibodies anti-Bcl-2 (lot 11o9905, Affinity Biosciences, Cincinnati, OH, USA), anti-Bax (Affinity, lot 44q6915), anti-PKCβ (Affinity, lot 69q9084), anti-Cyt-C (Affinity, lot 73c2522), anti-cleaved caspase-3 (Affinity, lot 15z0096), anti-phospho-Erk1/2 (Cell Signaling Technology, lot 24, Boston, USA), anti-ERK1/2 (Servicebio, lot LS193349, Wuhan, China), and anti-β-actin (Affinity, lot: 7) overnight at 4°C. The membranes were washed 3 times and incubated with secondary antibody HRP-goat anti-rabbit IgG (Affinity, lot 20000135) for 1 h. After washing 3 times, ECL reagents (Vazyme, Nanjing, China) were added for chemiluminescent imaging. The protein bands were quantitatively analyzed by an ImageJ software.
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Total protein was extracted from cells using RIPA buffer (Beyotime, Shanghai, China) in the presence of a protease inhibitor mixture (Pierce Chemical, Dallas, Texas, USA) and quantified using a BCA protein assay kit (Thermo Scientific, Waltham, USA). Western blotting was performed according to standard procedures. The following primary antibodies were used: antiperiostin (Proteintech, #66,4911lg, 1:3000), antiERK1/2 (CST, #9102, 1:1000), antiphospho ERK1/2 (CST, #4370, 1:1000), antiP38 (CST, #9212, 1:1000), antiphospho P38 (CST, #9215, 1:1000), and antiβactin (Affinity Biosciences, #AF7018, 1:1000).
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The supernatant protein was collected following the lysis of Huh7 cells. Protein samples were quantified using a BCA Kit. Protein samples were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (PVDF). At the end of transfer, blocking was performed using 5% nonfat milk for about 1 h at room temperature conditions. Blots were probed with anti-MEX3C (Santa, 398,440) and anti-β-Actin (Affinity, AF7018) antibodies, which were incubated overnight at 4 ° C on a shaker. The next day, the membranes were washed using Tris-buffered saline with Tween 20 (TBST). Subsequently, the membrane was next washed using TBST after binding with secondary antibodies for 1 h at room temperature. Finally, the amount of protein expression in the sample bands was detected using chemiluminescence.
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After different treatments, the BV-2 cells were washed with cold PBS twice, and the cells were fully lysed with RIPA lysate containing 1% protease inhibitor. The lysate was collected and centrifuged at 4 °C at 12,000 rpm for 25 min. Part of the supernatant was taken for protein-concentration determination, and the remaining supernatant was mixed with the loading buffer and boiled at 100 °C for 10 min. Fifty μg of protein were injected into the pore of 12% and 8% SDS polyacrylamide gel to isolate proteins with different molecular weights. The protein was then transferred at a constant voltage to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was immersed in TBST solution containing 5% skim milk and slowly shaken at room temperature for 1 h. Wash the protein band with TBST three times. Anti-β-actin (1:1000) (Affinity, Changzhou, China), Anti-COX2 (1:1000) (Bioss, Beijing, China), anti-NLRP3 (1:1000) (GeneTex, Southern California, CA, USA), and anti-Caspase-1 (1:500) (Wanleibio, Shenyang, China) antibodies were used to incubate the protein bands at 4 °C overnight. On the second day, protein bands were incubated with either goat anti-rabbit (1:10,000) or goat anti-mouse secondary antibody (1:10,000) (ZSGB BIO, Beijing, China) for 1 h. All protein bands were measured by ECL Western blot assay.
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The proteins were extracted with RIPA lysis buffer and PMSF; then, the protein quantification was conducted with a BCA protein quantification kit. Processed protein samples were collected for 8–10% SDS-PAGE electrophoresis and transferred on PVDF membranes. The membranes were blocked with 1× TBST containing 5% skimmed milk powder for 1 h at room temperature. After membrane washing, the membranes were incubated with the primary antibodies listed below at a temperature of 4 °C overnight: anti-TLR4 (1:1000, Protein-tech, Wuhan, China), anti-NF-κB (1:1000, Protein-tech, Wuhan, China), anti-MyD88 (1:1000, Protein-tech, Wuhan, China), anti-AQP1 (1:1000, Protein-tech, Wuhan, China), anti-HO-1 (1:1000, CST, Boston, MA, USA), anti-Nrf2 (1:1000, Protein-tech, Wuhan, China), and anti-β-actin (1:1000, Affinity, Changzhou, China). The membranes were then incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit/mouse antibodies (1:3000, protein-tech, Wuhan, China) for 1 h at 37 °C, and the membranes were rinsed 6 times with 1× TBST to remove any remaining antibodies (5 min every time). The images were captured using an Automatic Electrophoresis Gel Imaging System (Tanon 5200 Multi, Shanghai, China).
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Rats were euthanized by anesthetic over‐dose with isoflurane inhalation. The lumbar spinal cord (L3–5) were collected and homogenized to obtain protein extract for Western blot analysis. Protein extract was centrifuged at 16,000g for 15 min at 4°C.
The equal quantified proteins (50 μg) were denatured and loaded for separation by 10%–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, then transferred to the polyvinylidene fluoride membrane (Millipore). Membranes were blocked with 5% nonfat milk/TBST for 2 h at room temperature and probed with the following primary antibodies: rabbit anti‐IL‐6, anti‐IL‐6Rα, anti‐JAK2, anti‐pJAK2, anti‐STAT3, anti‐pSTAT3, anti‐MIF, anti‐Cox‐2, and anti‐β‐actin (1:1000; Affinity). Subsequently, the membranes were incubated with 1:10,000 horseradish peroxidase‐conjugated goat anti‐rabbit secondary antibody (Affinity) in 5% nonfat milk/TBST for 1 h at room temperature. Finally, proteins were visualized with ECL reagent (Affinity) and analysed by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
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We performed western blotting (WB) according to standard procedures. The following primary antibodies were used: anti-Hnrnpk (Abcam 39975, 1:5000), anti-Bax (Cell Signaling Technology 14796, 1:1000), anti-p21 (Abcam 109520, 1:1000), anti-Ihh (Abcam 52919, 1:1000), anti-Runx2 (Abcam 192256, 1:1000), anti-Mmp13 (Abcam 219620, 1:500), anti-Sox9 (Abcam 185966, 1:1000), anti-Hif1α (Cell Signaling Technology 36169, 1:250), anti-Gapdh (Proteintech 60004-1, 1:2000), anti-Pfkfb3 (Abcam 181861, 1:500), anti-Ldha (Proteintech 19987, 1:2000), and anti-β-Actin (Affinity AF7018, 1:2000). The following secondary antibodies were used: goat anti-rabbit IgG H&L (Abcam ab205718, 1:2000) and goat anti-mouse IgG H&L (Abcam ab205719, 1:2000). The Densitometry analysis of WB was exerted using software Image J (v 1.51). The original western blots were shown in the Supplementary material Western blots.
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Carbon monoxide was purchased from the Chengdu Xindu Jinnengda Gas Co. (China). sodium butyrate was purchased from the Shanghai Macklin Biochemical Technology Co. (China). The BCA protein assay kit was purchased from Solarbio (China). The anti-HDAC1, anti-NeuN, anti-mTOR, anti-p-mTOR, anti-P62, anti-Beclin1, anti-LC3B, anti-Bax, anti-Bcl-2, anti-β-actin primary antibodies, and HRP-labeled goat anti-rabbit secondary antibodies were purchased from Affinity (USA). CoraLite594-conjugated Goat Anti-Rabbit IgG (1:250) was purchased from Proteintech (USA).
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