Plates were rinsed twice with PBS before harvesting of the adherent cells. The collected cells were lysed with 1 mL of radioimmunoprecipitation assay (RIPA) lysis buffer (R0010; Solarbio, China) supplemented with 1 mM phenylmethyl sulfonyl fluoride (PMSF), 10 mM dithiothreitol (DTT), 40 μg/mL DNase I, and 1 μg/mL leupeptin, pepstatin, and aprotinin for 30 min on ice. Cell lysis was performed by centrifugation at 12,000 rpm for 30 min at 4°C. The supernatant was collected, and the protein concentration was measured using the bicinchoninic acid (BCA) protein assay kit. Equal amounts of protein were mixed with 1× SDS-PAGE loading buffer, boiled, and subjected to SDS-PAGE. The proteins in the gel were transferred onto 0.45-μm-pore polyvinylidene difluoride (PVDF) membranes and detected with specific or tagged antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. ImageJ was used to quantify the relative band intensities. The following antibodies were used: GFP-tagged monoclonal antibody (66002-1; Proteintech, IL, USA), Flag-tagged polyclonal antibody (20543-1; Proteintech), β-tubulin polyclonal antibody (10068-1; Proteintech), β-actin polyclonal antibody (20536-1; Proteintech), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody (10494-1; Proteintech), and ABHD16A/BAT5 polyclonal antibody (SRP08788; Saierbio, China).
Gapdh polyclonal antibody
Manufactured by Proteintech
Sourced in United States, China
The GAPDH polyclonal antibody is a lab equipment product designed for research purposes. It targets the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein, which is a widely expressed enzyme involved in glycolysis. This antibody can be used to detect and analyze GAPDH expression in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
β actin polyclonal antibody, by Proteintech (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Bca protein assay reagent kit, by Transgene (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Whole cell lysis buffer, by Keygen Biotech (1 mentions)
P ampk, by Cell Signaling Technology (1 mentions)
Lamp2, by Abcam (1 mentions)
P21 rabbit polyclonal antibody, by Proteintech (1 mentions)
P70 s6 kinase antibody, by Cell Signaling Technology (1 mentions)
Lc3b antibody, by Cell Signaling Technology (1 mentions)
Interleukin 6 (il 6), by Cell Signaling Technology (1 mentions)
Cd206, by Abcam (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Nanjing Chemical Reagent (1 mentions)
Cell lysis buffer, by Solarbio (1 mentions)
Hrp conjugated affinipure goat anti mouse igg, by Proteintech (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Licor scanner, by LI COR (1 mentions)
8 protocols using gapdh polyclonal antibody
1
Protein Extraction and Western Blot Analysis
2
Analysis of miR-29a Modulation on DPP4 Protein
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K1 cells of the miR-29a mimics group, the control mimics group, the miR-29a inhibitor group, and the control inhibitor group were collected after 48 h of incubation. Prechilled PBS was used to wash cells three times. Total protein in these cells was extracted by using whole cell lysis buffer (Nanjing KeyGen Biotech Co., Nanjing, China). Subsequently, the protein concentration was determined using a bicinchoninic acid assay (BCA) protein assay reagent kit (Beijing TransGen Biotech Co., Beijing, China). Then, 50 μg protein samples were subjected to 10% SDS-PAGE at 4°C for 2 h, followed by transferring to a polyvinylidene difluoride (PVDF) membrane by wet electrotransfer method at 4°C. The membrane was then blocked with 5% skim milk-tris-buffered saline-Tween 20 (TBST) for 2 h at room temperature. Rabbit anti-rat DPP4 primary antibody (1:1,000, Santa Cruz, Germany) was added for 12 h incubation at 4°C. After washing with TBST three times, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibody (1:2,000, Vector, USA) for 1 h at room temperature. GAPDH polyclonal antibody (1:1,000, Proteintech Group, Chicago, IL, USA) served as the control. Finally, blots were developed by a chemiluminescence detection system (ECL-Plus from Amersham, GE Healthcare UK Ltd, Little Chalfont, UK).
3
Cell Lysate Preparation and Western Blotting
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The culture medium was removed, and after washing the cells twice with PBS, they were harvested for further analysis. The preparation of cell lysates and Western blotting were performed by the method of Sun et al. [42 (link)]. Antibodies used were p16 INK4A (F-12) (Santa Cruz Biotechnology, sc-1661), P53 mouse monoclonal antibody (Proteintech, 60283-2-Ig), P21 rabbit polyclonal antibody (Proteintech, 27296-1-AP), LC3B antibody (Cell Signaling, 2575S), LAMP2 (Abcam, ab13524), AMPK (Cell Signaling, D63G4), P-AMPK (Cell Signaling, 2535S), p70 S6 Kinase Antibody (Cell Signaling, 9202S), p-p70 S6 Kinase Antibody (Cell Signaling, 9234S), and GAPDH polyclonal antibody (Proteintech, 10494-1-AP).
4
Protein Expression Analysis in Myocardial Tissue
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Fresh myocardial tissue was lysed by RIPA lysis buffer (Nanjing, China) and proteins were extracted. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The membrane was closed with 5% bovine serum albumin and then incubated with the appropriate primary antibody overnight at 4 °C, followed by washing with TBS-Tween and incubation with the appropriate secondary antibody at room temperature for 1 h. Spots were detected using an ECL system (Amersham, UK) and optical density was measured using ImageJ software. The following antibodies were used: iNOS (Cell Signaling Technology, 13120S), CD206 (Abcam, ab125028), (Abcam, ab28946), IL-6 (Cell Signaling Technology, 12912S), Stat3 (Cell Signaling Technology, 9139S), Phospho-Stat3 (Cell Signaling Technology, 9145S), GAPDH polyclonal antibody (Proteintech, No.10494–1-AP).
5
Protein Expression Analysis of Cultured Cells
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Cell lysis buffer (#R0010, Solarbio, China) was used to collapse the cells on the ice, phenylmethanesulfonylfluoride (PMSF, #P0100, Solarbio) was added in one hundredth of the volume of lysis buffer to restrain the degradation of protein. The protein concentration of lysates was quantified using the Enhanced BCA Protein Assay Kit (#P0010S, Beyotime, China). The western blot assay was strictly following the procedures of a previous publication21 (link). The primary antibodies included ALKBH5 Monoclonal antibody (#67811-1-Ig, Proteintech, China), VEGFA Monoclonal antibody (#66828-1-Ig, Proteintech, China), VAV1 Polyclonal antibody (#16364-1-AP, Proteintech, China), FGFR1 Monoclonal antibody (#60325-1-Ig, Proteintech, China), and GAPDH Polyclonal antibody (#10494-1-AP, Proteintech, China). The horseradish peroxidase (HRP)-conjugated affinipure goat anti-rabbit IgG (1:5000, SA00001-2, Proteintech, China) and HRP-conjugated affinipure goat anti-mouse IgG (1:5000, SA00001-1, Proteintech, China) served as corresponding secondary antibodies after incubation with primary antibody. The value of protein was calculated using the ImageJ software.
6
Quantifying STIM1 and Orai1 Proteins
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Western blot analysis was used to measure STIM1 and Orai1 protein expression in the LV myocardial tissues. Equal concentrations of proteins were separated according to their molecular weight using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred onto 0.02 mm polyvinylidene fluoride membranes (Millipore, United States), and blocked with 5% low-fat silk milk (Claix, France) for 3 h at room temperature. The membranes were subsequently incubated with STIM1 monoclonal rabbit anti-rat primary antibody (1:2500; Abcam, United States), Orai1 monoclonal rabbit anti-rat primary antibody (1:2500; Abcam, United States), and GAPDH polyclonal antibody (1:1000; Proteintech, United States) overnight at 4°C. Polyvinylidene fluoride membranes were washed thrice in Tris-buffered saline with Tween 20 and incubated with goat anti-rabbit secondary antibodies (1:1000; LI-COR, United States). Protein bands were detected using a LI-COR scanner (LI-COR, United States). Tris-buffered saline with Tween 20 was used for all antibody dilutions. Quantification of protein expression was achieved using Image J software (International Institute of Health, United States).
7
Protein Extraction and Western Blot Analysis
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Total proteins were extracted using a total protein extraction kit (Beyotime, Jiangsu, China). Western blot was performed according to the standard process and transferred to PVDF membranes. The primary antibodies were purchased from the Abcam and Proteintech, including CYP26A1 monoclonal antibody (Abcam, 1 : 5000), E-cadherin polyclonal antibody (Proteintech, 1 : 5000), N-cadherin polyclonal antibody (1 : 2000), Vimentin polyclonal antibody (1 : 5000), and GAPDH polyclonal antibody (1 : 10000).
8
Western Blot Analysis of Stem Cell Markers
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Cells were lysed with PhosphoSafe TM extraction reagent (Merck Millipore, Darmstadt, Germany) containing 1× proteasome inhibitor (Sigma). Thirty micrograms of protein was loaded onto 10% gradient NuPAGE gels (Novex, San Diego, CA, USA) for electrophoresis and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). Western blotting was performed using the primary antibodies glyceraldehyde 3-phosphate dehydrogenase (GAPDH) polyclonal antibody (1:5000 dilution in 5% milk, Proteintech), SPARC monoclonal antibody (1:500, Abnova, Taibei), SOX2 polyclonal antibody (Abnova), and OCT4 polyclonal antibody (Proteintech) (1:1000) and secondary goat anti-rabbit antibodies (1:4000, PerkinElmer, USA). Band intensities were analyzed using Quantity One 4.6.6 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
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