Total protein was extracted from cells with RIPA buffer (Sigma), protein lysates (15–30 μg) were separated on NuPAGE 4–12% Bis-Tris gels (Novex) and transferred to polyvinylidene difluoride membranes (Roche). Membranes were blocked and incubated overnight with primary antibodies: anti-CD44 (Cell Signaling, #3578 Danvers, MA), anti-vimentin (Abcam, ab92547), anti-phophorylated S6K (Cell Signaling, #9234), anti-phophorylated 4EBP-1 (Cell Signaling, #2855), anti-xCT (SLC7A11, Cell Signaling, #12691), anti-α-Tubulin, (Santa Cruz, #sc-8035), anti-β- Actin, (Cell Signaling), #4970 or anti-GAPDH antibodies (Santa Cruz Biotechnology, sc-32233). A secondary anti-rabbit (Cell Signaling) or anti-mouse immunoglobulin G antibody peroxidase conjugate (GE Healthcare) was detected using ECL Western Blotting Analysis System (Amersham Biosciences).
Anti gapdh antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom, China, Germany, Japan
The Anti-GAPDH antibody is a primary antibody used to detect the presence and quantity of the GAPDH protein in biological samples. GAPDH, or Glyceraldehyde 3-phosphate dehydrogenase, is a key enzyme involved in the glycolysis pathway and is commonly used as a loading control or reference protein in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (32 mentions)
Quantity one software, by Bio-Rad (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Polyvinylidene fluoride membrane, by Merck Group (7 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (7 mentions)
Bca protein assay kit, by Beyotime (7 mentions)
Protease inhibitor cocktail, by Roche (6 mentions)
Ripa buffer, by Beyotime (5 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Anti akt, by Cell Signaling Technology (4 mentions)
Immobilon p membrane, by Merck Group (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Chemidoc imaging system, by Bio-Rad (4 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (4 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (4 mentions)
Complete protease inhibitor cocktail, by Roche (4 mentions)
Ecl western blotting detection system, by GE Healthcare (4 mentions)
Gapdh, by Santa Cruz Biotechnology (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
298 protocols using anti gapdh antibody
1
Protein Extraction and Western Blotting
2
Antibody Characterization in Cell Biology
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Mouse monoclonal anti-cortactin (clone 4F11), rabbit polyclonal anti-CYFIP1 antibody, mouse monoclonal anti-ARP2/3 complex antibody (clone 13C9), and rabbit polyclonal anti-TGN46 were purchased from Merck Millipore (Darmstadt, Germany), mouse monoclonal anti-RAC1 and rabbit polyclonal anti-Sec13a were from ThermoFisher Scientific (Waltham, MA, USA), and the antibodies against phosphorylated ERK1/2 (Thr202/Tyr204), total-ERK1/2, total-PAK1, phospho-PAK1/2 (Thr423/Thr402), total p38 MAPK, and the anti-GFP antibody from Cell Signaling Technology Inc. (Danvers, MA, USA). The antibodies against EEA1 and LAMP1 were from BD Biosciences (San Jose, CA, USA). The polyclonal antibody for detecting GM130 was from R&D systems. GFP-Trap resin was purchased from Chromotek GmbH (Martinsried, Germany), the anti-Na+/K+-ATPase and the anti-GAPDH antibodies were from Santa Cruz Biotechnologies (Dallas, TX, USA). All secondary HRP- and Alexa Fluor-labeled antibodies were from ThermoFisher Scientific.
3
Protein Expression Analysis in PANC-1 Cells
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After the indicated treatment, the PANC-1 cells were washed and lysed and the proteins in the cell lysate were separated by centrifugation. The proteins were separated on 10% SDS-PAGE gel and then transferred to PVDF membranes (Bio-Rad) and immunoblotted with the mouse anti-E-cadherin (Cell Signaling) and anti-GAPDH antibodies (Santa Cruz Biotechnology). At last, the proteins were visualized using ECL-plus detection system (Pierce).
4
Kisspeptin Protein Expression Analysis
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The fresh hypothalamic tissues were separated and the total proteins were extracted using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.). The BCA method was used for protein quantification (Beijing Solarbio Science and Technology Co., Ltd.). Proteins were separated via 12% SDS-PAGE (20 µg per lane) and transferred onto a PVDF membrane (MilliporeSigma). After blocking with 5% skimmed milk for 2 h at room temperature, rabbit anti-rat kisspeptin polyclonal (1:50; cat. no. sc-15400; subtype of kisspeptin not available; Santa Cruz Biotechnology, Inc.) and anti-GAPDH antibodies (1:8,000; cat. no. AP0063; Bioworld Technology, Inc.) were added and the membranes were incubated overnight at 4˚C. After washing with 2.5% TBS-Tween 20 three times, the goat anti-rabbit IgG HRP-conjugated secondary antibody (1:80,000; cat. no. E030120; Beijing Merida Technology Co., Ltd.) was added. After incubation for 1 h at room temperature, the membrane was washed three times and then analyzed using the Chemiluminescence image analysis kit (Tanon Science & Technology Co., Ltd.). Image J analysis software (version 1.46r; National Institutes of Health) was used to analyze the average gray value.
5
Macrophage Phenotype Characterization in PDX
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Mouse RAW264.7 macrophages were obtained from American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (Gibco, Life Technologies). PDX was purchased from Cayman (Cayman Chemical, Ann Arbor, MI, USA). IL-6, TNF-α, MCP-1 and IL-10 (enzyme-linked immunosorbent assay, ELISA) kits were obtained from RayBiotech (RayBiotech Inc., Norcross, GA, USA). Carboxylate-modified yellowgreen (YG) microspheres were purchased from Invitrogen (Thermo Fisher scientific, Waltham, MA, USA). FITC-anti-F4/80, PE-anti-CD11b and PE-anti-CD206 were obtained from eBioscience (eBioscience, San Diego, CA, USA). Anti-Arg1 antibody was purchased from Abcam (Abcam, Cambridge, MA, USA). Anti-Ym1 antibody was purchased from Stemcell Technologies (Stemcell Technologies, Vancouver, Canada). Anti-PPAR-γ and anti-GAPDH antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
6
Western Blot Analysis of ACE2 Expression
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The experimental procedures for Western blotting were described previously [16 (link)]. Briefly, after treatment with CSF for 48 h, the cells were washed with PBS and lysed using RIPA buffer. Equal amounts of protein (20 μg) were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (EMD Millipore Corporation, Billerica, MA, USA). The membranes were blocked with 5% skim milk and incubated with primary antibodies overnight at 4 °C. The primary antibodies included anti-ACE2 (Abcam, Cambridge, MA, USA) and anti- GAPDH antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were washed with Tris-buffered saline containing Tween 20 and incubated with a secondary antibody for 1 h at room temperature. After washing, protein bands were detected using the ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA).
7
Quantifying Protein Levels in MDA-MB-231 Cells
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Total cell protein concentrations from MDA-MB-231 cells treated with apigenin, with and without TNFα co-treatment, was determined using a modified Bio-Rad “DC” protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Cell lysates were separated by electrophoresis on 10% SDS-polyacrylamide gels and then transferred to Immobilon-P PVDF membranes. Blots were blocked at 4°C overnight in 5% bovine serum albumin (Sigma, St. Louis, MO, USA) in Tris-buffered saline with 0.05% Tween 20 in PBS (PBST) and then incubated overnight at 4°C with mouse anti-human ERK1/2 and IKBKe affinity purified antibody (Cell Signaling, Danvers, Ma, USA). Membranes were washed with PBST and incubated overnight with anti-goat IgG-horseradish peroxidase (Santa Cruz Biotechnology, CA) in PBST overnight at 4°C. Protein loading was monitored in each gel lane by probing the membranes with anti-GAPDH antibodies (Santa Cruz, Ca, USA). Immunoblot images were obtained using a Flour-S Max Multimager (Bio-Rad Laboratories, Hercules, CA). Lane density data was acquired with Quantity One Software (Bio-Rad Laboratories, Hercules, and CA).
8
Western Blot Protein Analysis Protocol
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The samples were lysed using CelLytic M Cell Lysis Reagent (Sigma Chemical Co., St. Louis, MO) in the presence of a protease inhibitor (SERVA Electrophoresis GmbH, Heidelberg, Germany). The cell lysates were separated via 10% SDS-PAGE and electro-transferred to PVDF membranes (GE HealthCare, Piscataway, NJ). These membranes were subsequently incubated with anti-TM, anti-fibronectin, anti-ezrin, anti-vimentin, and anti-GAPDH antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Then, horseradish peroxidase-conjugated secondary antibodies (1:5000) were applied. Next, the protein bands were visualized using an enhanced chemiluminescence reagent (GE HealthCare, Piscataway, NJ) and detected using a VersaDoc 5000 imager (Bio-Rad Laboratories, Hercules, CA) [23 (link)–25 (link)].
9
Western Blot Analysis of ZKSCAN1 Protein
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Western blotting was performed according to standard methods as described previously (Luo et al., 2015 ) using anti‐ZKSCAN1 Abcam (Cambridge, MA, USA) and anti‐GAPDH antibodies Santa Cruz (Santa Cruz, CA, USA). After washing, the membranes were incubated with horseradish peroxidase‐conjugated goat anti‐mouse or anti‐rabbit secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) and visualized using enhanced chemiluminescence reagents (Forevergen Biosciences).
10
Antibody Characterization and Cell Line Authentication
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