The frozen wing samples were ground and transferred to centrifuge tubes with RIPA/PMSF (Solarbio, China). The tubes with comminuted samples were incubated in ice for 2 h. The whole divided protein was collected by centrifuging at 4°C for 10 min (12000×g) and adjusted to almost the same protein level. Soluble protein with 4× sample loading buffer was separated on 8–12% SDS-PAGE gel and electrophoresed at 4°C. The protein was transferred onto NC membrane (Millipore Corporation, Billerica, MA, USA) by wet transfer after electrophoresis, then the NC membrane was blocked with 5% fat-free milk in TBST. The MC1R antibody (ab 180776, Abcam, UK), ASIP antibody (ab151033, Abcam, UK), and β-actin antibody (ab8227, Abcam) were incubated at 4°C overnight. The membrane was washed 3 times using TBST buffer, then hybridized with HRP-conjugated goat anti-rabbit IgG for 2 h at room temperature. After washing 5 times with 1× TBST, the proteins of MCIR, ASIP, and β-actin were detected on the membrane with the ultra-sensitive horseradish from the catalase DAB color kit (Sangon Biotech, China). The bands on the membrane were scanned by a densitometric analysis system (Bio-Rad).
Nc membrane
Manufactured by Merck Group
Sourced in United States, Germany, China
NC membranes are porous membranes made of nitrocellulose material. They are designed for use in various laboratory applications, including filtration, blotting, and immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (20 mentions)
Ripa lysis buffer, by Beyotime (13 mentions)
Ripa buffer, by Beyotime (10 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (9 mentions)
Ripa buffer, by Thermo Fisher Scientific (8 mentions)
Bca protein assay kit, by Beyotime (8 mentions)
Bca kit, by Thermo Fisher Scientific (8 mentions)
Odyssey imaging system, by LI COR (7 mentions)
Gapdh, by Cell Signaling Technology (6 mentions)
Odyssey infrared imaging system, by LI COR (5 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (5 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (5 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
β actin, by Abcam (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Ab8227, by Abcam (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Ecl kit, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Image analyzer, by Bio-Rad (3 mentions)
231 protocols using nc membrane
1
Protein Expression Analysis of MC1R and ASIP
2
Western Blot Analysis of Kidney Proteins
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Kidney homogenate and podocytes were collected and lysed in cell lysis buffer (Beyotime, Haimen, China) with protease inhibitor cocktail and phosphatase inhibitor (both from Sigma-Aldrich) for protein extraction. Equal amount of protein lysates (30 μg) were separated by 10% serum dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and electrotransferred onto nitrocellulose (NC) membranes (Millipore, Billerica, MA, USA). After being blocked with 5% non-fat dry milk in PBS for 1 h, the membranes were probed with the primary antibodies against TLR4, phosphorylated-p65 (p-p65), p65, p-IκBα, IκBα, Cleaved Caspase-3, Bcl-2 and β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight, followed by incubated with a horseradish peroxidase-conjugated secondary antibody (Invitrogen) for 2 h at room temperature. Peroxidase-labeled protein bands were detected by enhanced chemiluminescence reagents (Millipore) and the protein intensity was quantified with Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).
3
Assessing GldM Expression in Complementation Strain
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To determine whether the complementation strain cYb2ΔgldM expressed GldM, whole-cell proteins of the wild-type strain Yb2, mutant strain Yb2ΔgldM, and complementation strain cYb2ΔgldM were extracted and separated by SDS-PAGE and then electrophoretically transferred onto nitrocellulose (NC) membranes (Millipore, Billerica, MA, USA). The membranes were blocked in phosphate-buffered saline (PBS) containing 5% nonfat milk, washed with PBS containing 0.05% Tween 20, and incubated overnight with the rabbit anti-rGldM polyclonal antibody. A horseradish peroxidase-conjugated goat anti-rabbit IgG polyclonal antibody (Bio-Rad Laboratories, Hercules, CA, USA) was then applied, and the specific bands were developed with the Basic Luminol Chemiluminescent Kit (S-Wb001), visualized using a Tanon 5200 automatic chemiluminescence image analysis system (Tanon, Shanghai, China), and quantified using ImageJ software (National Institutes of Health, Rockville, USA). A rabbit anti-TonB-dependent receptor antibody was used as the control for protein loading. The intensities of the protein bands were analyzed with Quantity One software (Bio-Rad Laboratories).
4
Protein Separation and Antigenicity Analysis
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The purified rSeCP and the crude and ES plerocercoid antigens were separated by SDS–PAGE (12% gel) and transferred onto nitrocellulose (NC) membranes (Millipore, USA) at 18 V for 35 min by semi-dry transfer cell (Bio-Rad, USA). Western blotting of rSeCP antigenicity was performed as described previously [25 (link),30 (link)].
5
Protein Extraction and Analysis
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Transplanted tumor tissue or cell protein were extracted with lysis buffer. The concentration of protein was determined by BCA assay kit (Beyotime, Shanghai, China). Protein samples were analyzed by SDS-PAGE using a 10%–12% polyacrylamide and transferred onto the NC membranes (Millipore, Billerica, MA, USA). Immune complexes were formed by incubation of primary antibodies for overnight at 4 °C, then followed conjugated second antibody for 1 h at 37 °C. Immunoreactive protein bands were detected with Tanton chemiluminescenc image analysis system (Shanghai, China), and the density of protein band was detected by Image J software.
6
Western Blot Analysis of PPAR-γ Expression
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Treated or transfected cells were collected at indicated time points, followed by the extraction of whole protein using RIPA buffer (Beyotime Biotechnology, Shanghai, China). Then, equal weight (40 µg) protein in each sample was separated by SDS-PAGE electrophoresis and then transferred to NC membranes (Millipore, Bedford, MA, USA). After blocking in 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against PPAR-γ or β-actin (Abcam, Cambridge, UK) overnight at 4℃. Subsequently, the membranes were probed with horseradish peroxidase-conjugated appropriate secondary antibody for 1 h at room temperature. Finally, specific protein signals in the membranes were detected using Clarity™ ECL Western Blotting Substrates (Bio-Rad, Hercules, CA, USA) and quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).
7
Protein Extraction and Western Blot
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Cells were lysed using RIPA buffer (Thermo Fisher, USA) enriched with 0.1% protease inhibitor, 1% phosphatase inhibitor, as well as 1% PMSF. Fractionation of the proteins was done on SDS-PAGE gel and then the proteins transfer-embedded onto NC membranes (Millipore, USA). Afterward, membranes were blocked with 5% skimmed milk in PBS for 2 h, followed by overnight incubation with anti-β-actin (Cell Signaling Technology, USA) and anti-DCTPP1 (Abgent Inc., USA) at 4°C. They were then washed thrice, 10 min each with PBST, and incubated for 1 h with indicated secondary antibodies.
8
Western Blot Protein Detection
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Cells were harvested and lysed in RIPA buffer with 1% phosphatase and protease inhibitors. Protein content was quantified by BCA protein quantification kits; the same amount (∼30 μg) of proteins was separated and transferred onto NC membranes (Millipore). After blocking membranes for 1 h at room temperature, the NC membranes were incubated with dilutions containing specific primary antibodies overnight at 4℃. Next day, the membrane was washed three times (10 min/each time) with 1 × TBST and incubated with HRP‐conjugated secondary antibodies for 1 h at room temperature. Finally, the membrane was washed three times (10 min/each time) again and visualized using the ECL exposure system (Thermo Fisher Scientific).
9
Immunofluorescence and Western Blot Analysis
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For immunofluorescence assay, cells were grown on glass slices and fixed in 4 % formaldehyde for 10 min, permeabilized through 0.3 % Triton X-100. Then the slices were blocked in goat serum for 15 min, 37°C and incubated overnight at 4°C with anti-E-cadherin (1:80, Bioworld, MN, USA), anti-N-cadherin (1:80, Bioworld, MN, USA). Samples were washed three times before incubated with goat TRITC labeled secondary antibody (1:70, Bioworld, MN, USA) at 37°C for 1 h. DAPI (Genview Inc, Shanghai, China) was used for counterstaining. Fluorescence was visualized with a microscope under ×400 magnification.
Total protein was extracted using RIPA Lysis Buffer (Beyotime, China) and PMSF (Sigma-Aldrich). The proteins were transferred to NC membranes (Millipore Corp, MA USA) using the TransBlot System (Bio-Rad, CA, USA). The membranes were blocked in 5% w/v non-fat milk in TBS and incubations were performed overnight at 4°C. The membranes were then washed using TBST and incubated with secondary antibodies (1:10000, IRDye Goat IgG, LI-COR Bioscience, NE USA) for 1h at room temperature. Protein staining was detected using the Odyssey Imaging System (LI-COR Biosciences, NE USA). The following primary antibodies were used: GAPDH (1:10000, Proteintech Group, Chicago USA), ABCC1 (1:100, Abcam, ab24102), ABCC2 (1:100, Abcam, ab3373), P-gp1 (1:2000, Abcam, ab129450).
Total protein was extracted using RIPA Lysis Buffer (Beyotime, China) and PMSF (Sigma-Aldrich). The proteins were transferred to NC membranes (Millipore Corp, MA USA) using the TransBlot System (Bio-Rad, CA, USA). The membranes were blocked in 5% w/v non-fat milk in TBS and incubations were performed overnight at 4°C. The membranes were then washed using TBST and incubated with secondary antibodies (1:10000, IRDye Goat IgG, LI-COR Bioscience, NE USA) for 1h at room temperature. Protein staining was detected using the Odyssey Imaging System (LI-COR Biosciences, NE USA). The following primary antibodies were used: GAPDH (1:10000, Proteintech Group, Chicago USA), ABCC1 (1:100, Abcam, ab24102), ABCC2 (1:100, Abcam, ab3373), P-gp1 (1:2000, Abcam, ab129450).
10
Far-Western Blot Analysis of rTsCTL Binding
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The binding of rTsCTL and IEC was investigated by far-Western blot analysis as previously described [16 (link)]. In brief, soluble IEC proteins were first separated by SDS‒PAGE and then transferred to NC membranes (Millipore, USA). The membrane was cut into strips, blocked with 5% skim milk at 37 °C for 2 h, and then incubated with 20 μg/mL rTsCTL for 2 h at 37 °C. Following washes with PBST, strips were probed at 37 °C for 1 h with anti-rTsCTL serum (1:100) as the primary antibody and HRP-anti-mouse IgG (1:10 000; Southern Biotech) as the secondary antibody [52 (link)]. After washing, colouration was developed using 3-amino-9-ethylcarbazole (AEC, Solarbio, China), and the protein bands were analysed by AlphaView software [18 (link), 53 (link)].
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