The total proteins were extracted from U-CH1 and U-CH2 cell lines using protein lysates (Beyotime, China). The proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. After blocking with BSA, the membranes were incubated with primary antibodies against NRP1 (Abcam, United States) and internal reference GAPDH (Beyotime, China). Then, the bands were treated with secondary antibody and were developed using chemiluminescence substance (Beyotime, China).
Protein lysate
Manufactured by Beyotime
Sourced in China
Protein lysate is a solution obtained by lysing cells or tissues to extract their intracellular proteins. It contains a complex mixture of proteins, enzymes, and other biomolecules that can be used for various analytical and experimental purposes.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pvdf membrane, by Beyotime (2 mentions)
Anti actin, by Beyotime (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Ab32445, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Ab115777, by Abcam (1 mentions)
Ab179800, by Abcam (1 mentions)
Ab133739, by Abcam (1 mentions)
Ab207297, by Abcam (1 mentions)
Ab178945, by Abcam (1 mentions)
Ab13847, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab222494, by Abcam (1 mentions)
Wb reagent, by Thermo Fisher Scientific (1 mentions)
Ab13556, by Abcam (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Topolyvinylidene fluoride membranes, by Merck Group (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
15 protocols using protein lysate
1
Western Blot Analysis of NRP1 Protein
2
Immunoblotting Analysis of NF-κB Pathway
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BMMs were seeded on the polyacrylamide hydrogels in the 6-well plates at density of 1.5 × 106 cells/well for 24h. Protein was extracted with protein lysates (Beyotime, China), the concentrations were measured using the BCA Protein Assay Kit (Beyotime, China). Equal amounts of total protein from cell lysates were electrophoresed in polyacrylamide hydrogels and transferred to nitrocellulose (NC) membrane from the polyacrylamide hydrogels. The nitrocellulose membranes were incubated with primary antibody (anti-IκB, NIK, p65, pi-p65, pi-IκB, Abcam, USA; anti-actin, Beyotime, China) with slight shaking at 4 °C for overnight. The NC membranes were washed and incubated horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG, goat anti-mouse IgG, Beyotime, China) for 1h at room temperature, signals were detected by chemiluminescence. Protein bands were showed and semi-quantified by Image Lab software (Bio-Rad, USA).
3
Molecular Mechanisms in MCAO Rats
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After the brain tissues and cells of MCAO rats were treated, we removed the medium. Then, the total proteins were isolated with protein lysates (Beyotime Biotechnology, Shanghai, China). Next, 50 μg of total protein was loaded onto a 12% polyacrylamide gel for 100 V electrophoresis for 2 h and transferred to polyvinylidene fluoride (Millipore, Bedford, MA, USA) membranes. At RT, 5% skim milk was used to block the membranes for an hour. TBST was used to wash them 3 times (10 min each time). Then, the membranes were incubated overnight (4°C) with anti-iNOS (1:1,000, ab178945, Abcam), anti-COX2 (1:1,000, ab179800, Abcam), anti-Bad (1:1,000, ab32445, Abcam), anti-Bax (1:1,000, ab32503, Abcam), anti-Caspase3 (1:1,000, ab13847, Abcam), anti-TREM1 (1:1,000, ab104413, Abcam), anti-TLR4 (1:1,000, ab13556, Abcam), anti-MyD88 (1:1,000, ab133739, Abcam), anti-NF-κB (1:1,000, ab207297, Abcam), anti-p-NF-κB (1:1,000, ab222494, Abcam), and anti-β-actin (1:1,000, ab115777, Abcam). Subsequently, the membranes were rinsed with TBST. The anti-rabbit secondary antibody labeled by horseradish peroxidase (HRP) (concentration: 1:300) was applied for a 1-h incubation at RT. TBST was used to wash the membranes 3 times (10 min/wash). Ultimately, Western blotting (WB) reagent (Invitrogen) was utilized for color imaging. ImageJ 1.44 software was employed for density detection.
4
Striatum Inflammatory Factors Quantification
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After anesthetization, the rats were killed by decapitation to acquire the lesioned striatum 1 h after the final drug administration. Each 100 μg striatal tissue was dissociated in 100 μl protein lysate (Beyotime, Shanghai, China) with the addition of protease inhibitor (Beyotime, Shanghai, China) at a ratio of 1:100. After tissue grinding, the mixed liquid was centrifugated at 12,000 rpm, 4°C for 10 min. The supernatant was collected for assay of TNF-α, IL-1β, IL-6, and iNOS by using ELISA kits (IL-1β and IL-6, Abcam, Shanghai, China) (TNF-α and iNOS, Elabscience, Wuhan, China) following the manufacturer’s instructions, respectively. Optical density was obtained at 450 nm using a microplate reader within 15 min of stop solution addition.
5
Protein Extraction and Western Blot Analysis
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Total proteins in each group of cells were
extracted using RIPA (radioimmunoprecipitation) protein
lysate (Beyotime, Shanghai, China). The extracted
proteins were separated using a 12% sodium
dodecyl sulphate-polyvinylidene polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred on to
polyvinylidene fluoride membranes (Millipore, USA).
Subsequently, the membranes were immersed in 5%
skim milk for 2 hours. Primary antibodies were incubated
for overnight incubation at 4°C. The next day,
the membranes were incubated with horse radish peroxidase
(HRP)-labeled secondary antibody for 2 h.
Bands were exposed using electrochemiluminescence (ECL) reagent. Glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) was served as the internal control.
extracted using RIPA (radioimmunoprecipitation) protein
lysate (Beyotime, Shanghai, China). The extracted
proteins were separated using a 12% sodium
dodecyl sulphate-polyvinylidene polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred on to
polyvinylidene fluoride membranes (Millipore, USA).
Subsequently, the membranes were immersed in 5%
skim milk for 2 hours. Primary antibodies were incubated
for overnight incubation at 4°C. The next day,
the membranes were incubated with horse radish peroxidase
(HRP)-labeled secondary antibody for 2 h.
Bands were exposed using electrochemiluminescence (ECL) reagent. Glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) was served as the internal control.
6
Western Blot Analysis of DPSCs
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DPSCs were inoculated into a 6-well plate (Corning) at a density of 1 × 105 cells per well. Once the fusion reached 100%, the cells were harvested and subjected to treatment with protein lysate (Beyotime, Shanghai, China) and PMFS (Beyotime, Shanghai, China). The protein concentration was determined using the BCA kit (Beyotime, Shanghai, China), and the protein was denatured by steaming at 100 °C for 8 min. An equal amount of total protein was loaded onto the prepared gel plate, and the membrane was sealed with 5% skimmed milk powder after transfer. The primary antibody was incubated overnight at 4 °C with agitation(Table 2 ). Subsequently, it was combined with the secondary antibody to visualize the immunoblotting bands. A marker (Epizyme, Shanghai, China) can be utilized as a reference for determining the molecular weight position of the target protein.
Antibody list
| Antibody | Manufacturers | Dilution rate |
|---|---|---|
| NRG-1 | Santa | 1:5000 |
| F-actin | Proteintech | 1:50000 |
| β3-tubulin | Proteintech | 1:50000 |
| NSE | Proteintech | 1:50000 |
| NF-κB | Proteintech | 1:50000 |
| P38 MAPK | Proteintech | 1:50000 |
| GAPDH | Proteintech | 1:50000 |
7
Western Blot Analysis in HeLa Cells
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48 h after the transfection of HeLa cells, the culture medium was removed. After PBS rinsing, the cells were lysed with protein lysate (Beyotime, China), and the total proteins were collected. Then the protein concentration was detected by BCA (Beyotime, China) protein quantitative kit. After adding proper amount of SDS loading buffer (Beyotime, China), denaturation was carried out in boiling water at 100°C for 5 min. Then 12% SDS-PAGE electrophoresis (Beyotime, China) was performed. Protein bands were transferred to PVDF membranes (Beyotime, China) by western transmembrane system. Subsequently, the PVDF membrane was sealed in 5% skim milk powder (Beyotime, China) and incubated for 4 h. After washing with TBST (Beyotime, China), the PVDF membrane was incubated with primary antibody at 4°C overnight. After washing, the membrane was incubated with the secondary antibody at room temperature for 60 min. Finally, the PVDF membrane was stained with western TMB substrate (Beyotime, China; P0211) and scanned. The antibodies used were β-actin (Beyotime, China; AF5003), TYMS (ABCAm, United States, ab108995) and horseradish peroxidase labeled goat anti-rabbit IgG (Beyotime, China; A0208).
8
Western Blot Analysis of Protein Signaling
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RASFs in each group were digested with protein lysate (Beyotime Biotechnology, Shanghai, China) to extract total protein. Bicinchoninic acid assay (BCA) kit (KeyGEN BioTECH, Nanjing, China) was used for protein quantitation. Firstly, the proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then, proteins were transferred to nitrocellulose membranes and sealed with 5% skim milk for 2 h. Diluted primary antibodies (PIK3R2: Santa Cruz Biotechnology, USA; PI3K: Santa Cruz Biotechnology, USA; AKT: Cell Signaling, USA; phospho-AKT: Cell Signaling, USA; β-actin: Cell Signaling Technology, USA) were added into the membranes, and the mixture was incubated at 4°C overnight. Then, the membranes were washed once with PBS twice with tris-buffered saline tween-20 (TBST). Horseradish peroxidase-labeled secondary antibodies were added and the mixture was incubated at room temperature for 2 h. After the membranes were washed, electrogenerated chemiluminescence (ECL) was used to develop the films, which were rinsed with pure water and left to dry. Scanning was used to record them.
9
Western Blot Analysis of NSC Proteins
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Protein lysate (Shanghai Beyotime Biotechnology Co., Shanghai, China) was added to the NSCs and frozen brain tissues to extract total protein. The Bradford method (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to assess protein quality. Total protein (50 μg) was then used for 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) electrophoresis and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk at 37°C for 1 hr, the membranes were incubated with rabbit anti-mouse NRSF (ab21635,1: 500), MOR (ab1009,1: 700), DOR (ab83775, 1: 1,000), Bcl-2 (ab32124,1: 1,000), Bax (ab32053, 1: 1,000), and β-actin (ab8227, 1: 1,000) monoclonal antibodies (Abcam, Inc., Cambridge, MA, USA) overnight at 4°C. Membranes were then rinsed with tris-buffered saline Tween-20 (TBST) 3 times for 5 min and horse radish peroxidase (HRP) labelled goat anti-rabbit second antibody (ab20272, 1: 4000; Abcam, Inc., Cambridge, MA, USA) was added. The membrane was incubated for 2 hrs at room temperature and then rinsed. Relative protein expression was quantified by comparing the gray value of the target band to the internal reference band.
10
Protein Expression Analysis of PDL Cells
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Total protein was obtained from PDL-derived cells by the protein lysate (Beyotime, China), and centrifuged with 12000 rpm for 10 min. Protein samples were then electrophoresed on 10% SDS-PAGE gel, transferred onto PVDF membranes (Millipore, USA), which was blocked with 5% non-fat milk. The primary antibody was incubated overnight at 4 °C and the secondary antibody was incubated for 1 h at room temperature. ECL solution was then prepared, and the bands on the membranes were scanned. The following primary antibodies were TGFβR2 (Abcam, UK), FGFR2 (Beyotime, China), osteopontin (OPN; Abcam, UK), osteocalcin (OCN; Abcam, UK), Runt-related transcription factor-2 (Runx2; Abcam, UK) and GAPDH (proteintech, USA).
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