Proteins were extracted from neuroblastomas cells, and then separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (PVDF). After blocked with 5% nonfat milk in TBST for 2 hours, the membrane was incubated with primary antibodies. Membranes were washed three times and incubated with the horseradish peroxidase-conjugated second antibodies. The signals were captured by the ECL reagent (Beyotime) and visualized by western blotting detection instruments (Clinx Science). Mouse anti-TAZ (560235; BD Biosciences), mouse anti-YAP (sc-101199, 1:200), goat anti-CTGF (sc-14939, 1:200) from Santa Cruz company, rabbit anti-PDGF-β (E1A0240-1, 1:1000) from EnoGene, and mouse anti-GAPDH (AG019, 1:1000) from Beyotime, cell cycle regulation antibody sampler kit #9932 from Cell Signaling Technology were used as primary antibodies. HRP-labeled goat anti-mouse IgG (H + L) (A0216, 1:5000) and goat anti-rabbit IgG (H + L) (A0208) were used as secondary antibodies which purchased from Beyotime.
Mouse anti gapdh
Manufactured by Beyotime
Sourced in China, United Kingdom, United States
The Mouse anti-GAPDH is a primary antibody that specifically recognizes the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein, which is a widely expressed and highly conserved enzyme involved in the glycolytic pathway. This antibody can be used for the detection and quantification of GAPDH in various sample types through techniques such as Western blotting, immunohistochemistry, or ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Hrp labeled goat anti mouse igg h l, by Beyotime (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit igg h l, by Beyotime (1 mentions)
Cell cycle regulation antibody sampler kit 9932, by Cell Signaling Technology (1 mentions)
Mouse anti taz, by BD (1 mentions)
Mouse anti yap sc 101199, by Santa Cruz Biotechnology (1 mentions)
Ecl reagent, by Beyotime (1 mentions)
Western blotting detection instruments, by Clinx (1 mentions)
A0216, by Beyotime (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Ipsen (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
A0208, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Biosharp (1 mentions)
Gapdh, by Bio-Rad (1 mentions)
Image labtm software, by Bio-Rad (1 mentions)
Mouse anti e cadherin, by BD (1 mentions)
Rabbit anti histone h3, by Cell Signaling Technology (1 mentions)
Rabbit anti vimentin, by Cell Signaling Technology (1 mentions)
Rabbit anti e cadherin, by Cell Signaling Technology (1 mentions)
15 protocols using mouse anti gapdh
1
Profiling Neuroblastoma Protein Signatures
2
MAML3 Protein Detection by Western Blot
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Cells were lysed in RIPA buffer (Beyotime, China) with 1% PMSF (Biosharp, China). The protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Epizyme, China) and transferred onto PVDF membrane (Millipore, USA). Primary antibodies were applied at 4 °C overnight and HRP-conjugated secondary antibodies were applied for an hour at room temperature. The immunocomplexes were detected with ECL Western Blotting Substrate (NCM Biotech, China), visualized with Tanon (5200multi 4600SF, Tanon, USA). GAPDH was used as the internal control. Primary Antibodies included rabbit antiMAML3 (1:500, Biorbyt, UK), mouse antiGAPDH (1:20000, Beyotime, China). Secondary antibodies (A0208 and A0216, Beyotime, China) were diluted in 1:1000.
3
Western Blot Analysis of P53 and GAPDH
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Protein lysates from cells were prepared in lysis buffer and centrifuged at 12, 000 rpm at 4 °C. Western blot was performed according to a previously described procedure 28 (link). The primary antibodies used were mouse anti-P53 (Beyotime, China) and mouse anti-GAPDH (Beyotime, China). The secondary antibody used was HRP-labeled goat anti-mouse IgG (H+L) (Beyotime, China). The expression of each band was quantitatively analyzed using the Image Lab TM Software (Bio-Rad, USA) and normalized to the expression of GAPDH in the same lane.
4
Cochlear Sensory Epithelium Protein Analysis
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Proteins were extracted from five pure sensory epithelia of apex and mid-apex cochlear explants that were isolated by removing the surrounding non-sensory epithelium. Western blotting was performed as described previously (Lu and Corwin, 2008 (link)). The following antibodies were used: mouse anti-E-cadherin (BD Biosciences, 1:2,500 dilution), anti-P120-catenin (Santa Cruz, 1:500 dilution), and mouse anti-GAPDH (Beyotime, China, 1:1,000 dilution). Proteins were detected using the Image Quant LAS 1040 detection system (GE Healthcare, Piscataway, NJ, USA). The band intensity was measured and normalized against the intensity of the GAPDH band measured from the same lane using ImageJ.
5
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Total protein samples were (30μg) separated by SDS polyacrylamide gel and then transferred to PVDF membrane. After blocking with 5% (W/V) nonfat-dried milk, membranes were incubated with primary antibodies at 1:1000 dilution at 4°C overnight, followed by incubation with the appropriate HRP-conjugated secondary antibodies (ZSGB-BIO) at 1:10000 dilution. The immune complexes were detected by an enhanced chemiluminescence system and exposed on Kodak X-ray films. Primary antibodies used are as follows: mouse anti-GAPDH (Beyotime), rabbit anti-MMP-2, rabbit anti-MMP-9, rabbit anti-E-cadherin, rabbit anti-vimentin, rabbit anti-β-catenin, rabbit anti-Histone H3 (Cell Signaling Technology), mouse anti-β-tubulin (Sigma-Aldrich), rabbit anti-snail and rabbit anti-twist (Zen Bioscience).
6
LPS-Induced Autophagy in 16HBE Cells
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Immortalized bronchial epithelial cell line 16HBE cells were obtained from Procell Technology (Wuhan, China). They were cultured in RPMI 1640 (Gibco) at 37°C with 5% CO2 and cocultured with 10% foetal bovine serum (Biological Industries). According to instruction, we dissolve the LPS powder into different concentrations and stimulate 16HBE for 12 hours.
Antibodies used were rabbit anti-LC3B (Cell Signaling Technology, 3868), rabbit anti-SQSTM1/p62 (Cell Signaling Technology, 8025), and mouse anti-GAPDH (Beyotime, AF5009). Lipopolysaccharide (Sigma-Aldrich, L4391), DAPI (Solarbio, C0065), and RAPA (Sigma-Aldrich, 553210) were used in this study.
Antibodies used were rabbit anti-LC3B (Cell Signaling Technology, 3868), rabbit anti-SQSTM1/p62 (Cell Signaling Technology, 8025), and mouse anti-GAPDH (Beyotime, AF5009). Lipopolysaccharide (Sigma-Aldrich, L4391), DAPI (Solarbio, C0065), and RAPA (Sigma-Aldrich, 553210) were used in this study.
7
Quantitative Protein Detection Protocol
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Cells were collected and directly lysed in lysis buffer containing RIPA Buffer (Beyotime) with PMSF (Sigma) and phosphatase inhibitor (Roche). Tissues were washed by cold PBS and then homogenized by IKA T10 homogenizer in lysis buffer. Proteins were quantified using Pierce BCA Protein Assay Kit (Thermo Scientific). Equal amounts of proteins were loaded for immunoblotting. Proteins were electroblotted to PVDF membranes; then, PBS with QuickBlot (Beyotime) was used to block membranes. Antibodies used were rabbit anti‐KLF3 (in‐house), goat anti‐KLF3 (Abnova, Cat. #PAB6147), mouse anti‐GAPDH (Beyotime, Cat. #AF0006), mouse anti‐β‐ACTIN (Biodragon, Cat. #B1029), mouse anti‐β‐TUBULIN (Biodragon, Cat. #B1031), mouse HSP90β (Beyotime, Cat. # AF0192), rabbit anti‐CKM (ProteinTech, Cat. #18712‐1‐AP) and rabbit anti‐MYL1 (ProteinTech, Cat. # 15814‐1‐AP). Uncropped Western blotting images are provided in Figure S7 .
8
Western Blot Analysis of PCBP1 Protein
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The cells were washed in ice-cold phosphate-buffered saline (PBS) and lysed in Cell Lysis Buffer for Western and IP (P0013, BEYOTIME) in the presence of 1mM PMSF (AR1192, BOSTER) and 1% Protease Inhibitor Cocktail (P1005, BEYOTIME). The protein concentrations were measured with the BCA assay Kit (AR1189, BOSTER) and 40 μg proteins were diluted in 5 × SDS-PAGE Loading Buffer (AR1112, BOSTER) at 95°C for 10 min. Subsequently, the samples boiled were resolved on the artificial 4–12% SDS-PAGE gel and the proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk dissolved in TBST for 2 h at room temperature. Primary antibodies for immunoblotting were as follows: rabbit anti-PCBP1 (1:2000, BOSTER A02636-1) and mouse anti-GAPDH (1:1000, BEYOTIME AG019). Membranes were subsequently washed in TBST and then incubated with horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG (H + L) (1:5000, BOSTER BA1056). Ultimately, membranes were imaged with the ultra-sensitive ECL chemical luminescence ready-to-use kit (BOSTER AR1197) using Azure c600 (AZUREBIOSYSTEMS). The corresponding protein bands were normalized to GAPDH band density using Fiji.
9
Western Blot Analysis of NFAT5 and NPEPPS
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The total protein was extracted from the PK15 cells using cell lysis buffer for Western blotting (Beyotime, China) with PMSF (Beyotime). The protein concentration of the cell lysate was quantified using a BCA kit (Beyotime), and 40 µg of each protein sample were separated on SDS-PAGE 8% gels and blotted onto a polyvinylidene fluoride (PVFD) membrane (Millipore, USA).The membranes were blocked for 2 h at room temperature in Western Blocking Buffer (Beyotime) and then incubated overnight at 4 °C with rabbit polyclonal antibodies against NFAT5 and NPEPPS (Abcam, USA) at a dilution of 1:1000. To normalize the protein loading, the PVDF membranes were simultaneously incubated with the mouse anti-GAPDH (Beyotime) monoclonal antibody at a dilution of 1:1000. The membranes were washed three times with TBST buffer and then incubated with HRP-conjugated secondary antibodies that were diluted 1000 times at room temperature for 1 h. Finally, the immunoreactive bands were visualized using a DAB Horseradish Peroxidase Color Development Kit (Beyotime).
10
Protein Expression Analysis of T Cells
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A total of 2 × 106 T cells were incubated with 200 μL of RIPA buffer system (Beyotime, Shanghai, China) for 10 minutes. The cell lysate was centrifuged to collect proteins, which were separated through SDS-PAGE, after which Western blot analysis was performed. The primary antibodies for CD34, CD3ζ, and GAPDH included rabbit anti-CD34 (CST, USA), mouse anti-CD3ζ (Abeam, UK), and mouse anti-GAPDH (Beyotime). The corresponding secondary antibodies were horseradish peroxidase (HRP) labeled goat antirabbit IgG (Beyotime) and goat antimouse IgG (Beyotime).
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