Total cell extracts were subjected to 12% SDS-PAGE and separated proteins transferred to polyvinylidene fluoride membrane. Western blot was performed using standard protocols described previously [46 (link)]. Rabbit anti-DEK, anti-E-cadherin, anti-vimentin, anti-MMP2, and anti-MMP9 antibodies were purchased from Proteintech (Chicago, IL, USA). Mouse anti-GAPDH antibody was obtained from Chemicon (Temecula, CA, USA).
Mouse anti gapdh antibody
Manufactured by Merck Group
Sourced in United States, United Kingdom
The Mouse anti-GAPDH antibody is a primary antibody that specifically binds to the GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) protein. GAPDH is a housekeeping gene commonly used as a loading control in various biological assays.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
C digit blot scanner, by LI COR (3 mentions)
Ecl plus system, by GE Healthcare (3 mentions)
Transfer apparatus, by Bio-Rad (3 mentions)
Las 3000 mini, by Fujifilm (2 mentions)
Goat anti col1a1, by Santa Cruz Biotechnology (2 mentions)
Amersham imager 600 system, by GE Healthcare (2 mentions)
Ecl substrate, by Merck Group (2 mentions)
Mouse anti il 33 antibody, by Abcam (2 mentions)
Masson s trichrome staining kit, by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Rat anti cd62p, by BD (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Coomassie plus protein assay reagent, by Thermo Fisher Scientific (2 mentions)
Supersignal west femto maximum sensitivity substrate kit, by Thermo Fisher Scientific (2 mentions)
50 protocols using mouse anti gapdh antibody
1
Protein Expression Analysis by Western Blot
2
Protein Extraction and Western Blot Analysis
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Kidney extracts were lysed in lysis buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton X-100, 0.3% NP40, 100 μM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 100 μM Na3VO4, and 1 mM protease-inhibitor cocktail (Sigma)]. Protein concentration was determined by the BCA method (Pierce). Tissue protein extracts (30 μg/lane) were separated on 8–12% polyacrylamide-SDS gels under reducing conditions. Samples were then transferred onto PVDF membranes (Bio-Rad), blocked in TBS with 0.05% Tween-20 and 5% nonfat dry milk, and then incubated overnight at 4°C with the primary antibodies and subsequently incubated with peroxidase-conjugated IgG (Amersham) and developed by ECL chemiluminescence (GE Healthcare). Autoradiographs were scanned using the GS-800 Calibrated Densitometer (Quantity One, Bio-Rad). Primary antibodies were affinity purified anti-mouse/human/rat Foxp3 (1 : 1000) (e-bioscience: 14-4774), NGAL (1 : 500) (Santa Cruz, sc-18698). The efficacy of protein loading and transfer to membranes was assessed by incubation with mouse anti-GAPDH antibody (1 : 5000) (Chemicon: MAB374). IL-17A levels were analyzed with an ELISA kit from eBioscience.
3
Western Blot Analysis of Autophagy
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Flag was detected using an anti-Flag M2 HRP antibody (1:2000) (Sigma-Aldrich, St. Louis, MO). The phosphospecific Beclin 1 S90 antibody was produced by PhosphoSolutions (Aurora, CO). Briefly, synthetic peptides corresponding to phosphorylated and dephosphorylated S90 of Beclin 1 were injected to two rabbits and sera were purified using a phosphopeptide affinity column followed by a dephosphopeptide affinity column. The phosphospecific Beclin 1 S90 antibody was used at a concentration of 1:500. Beclin 1, HSP27, p-HSP27, Actin, MK2, p62, LC3, TOM20, PDI, and GAPDH were detected using a rabbit or goat anti-Beclin 1 antibody (Santa Cruz Biotechnology, Dallas, TX, 1:1000 dilution), a rabbit anti-HSP27 antibody (Santa Cruz Biotechnology, 1:200 dilution), a rabbit anti-p-HSP27 antibody (Santa Cruz Biotechnology, 1:200 dilution), an anti-β-Actin HRP antibody (Santa Cruz Biotechnology, 1:2000 dilution), a rabbit anti-MK2 antibody (Cell Signaling Technology, Beverly, MA; 1:1000 dilution), a mouse anti-p62 antibody (Abnova, Walnut, CA; 1:2000 dilution) a rabbit anti-LC3 antibody (Novus Biologicals, Littleton, CO; 1:1000 dilution), a rabbit anti-TOM20 antibody (Santa Cruz Biotechnology; 1:1000 dilution), a rabbit anti-PDI antibody (Cell Signaling Techology; 1:1000 dilution), and a mouse anti-GAPDH antibody (Chemicon International, Temecula, CA; 1:000 dilution).
4
Gas6-Induced Axl Phosphorylation Assay
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Recombinant human Gas6 (885-GS, R & D Systems, Minneapolis, MN, USA) was used to stimulate H1299 cells. For detecting phosphorylation, cell extracts were prepared by lysing the cells in buffer containing 1 mM EDTA, 5 mM NaF, 0.5 mM sodium orthovanadate, 1 mM dithiothreitol (DTT), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and protein inhibitor cocktail (Nacalai Tesque, Kyoto, Japan). Lysates from whole cells were separated by 7.5% or 10%SDS-PAGE. Proteins were then transferred onto nylon membranes (Amersham, Buckinghamshire, UK) and incubated with mouse anti-α-SMA antibody (clone 1A4, Sigma-Aldrich), mouse anti-vimentin antibody (vim3B4, Dako), rabbit anti-PDGFR-α antibody (#3164, Cell Signaling Technology), rabbit anti-PDGFR-β antibody (#3169, Cell Signaling Technology), goat anti-Axl antibody (AF154, R&D Systems), rabbit anti-phospho Axl antibody (AF2228, R&D Systems), goat anti-Gas6 antibody (AF885, R&D Systems) or mouse anti-GAPDH antibody (Chemicon, Temecula, CA, USA) according to the manufacturers’ protocols. Proteins were detected with horseradish peroxidase-conjugated anti-rabbit, anti-goat, or anti-mouse secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) and ECL reagents (GE Healthcare, Milwaukee, WI, USA). The blots were scanned using an imaging densitometer LAS-3000 mini (Fujifilm, Tokyo, Japan).
5
Western Blot Analysis of E2A Protein
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Isolated bone marrow cells were washed in 1X PBS and homogenized in lysis buffer containing 50mM TrisCl pH7.5, 150mM NaCl, 1mM sodium pyrophosphate, 1mM NaF, 1mM benzamidine, 5mM sodium orthovanadate, and 0.5% (v/v) NP-40. Lysis buffer was supplemented with protease inhibitor cocktail (Sigma) prior to homogenization. Protein homogenates were loaded onto 10% SDS PAGE Gel and transferred overnight in 4°C at 25 mV onto a nitrocellulose membrane. After blocking, purified mouse anti-human E2A antibody (BD Pharmingen) was applied at dilution of 1:400 in 1% milk solution overnight in 4°C. Mouse anti-GAPDH antibody (Sigma) was applied at dilution 1:10,000 in 1% milk solution overnight in 4°C.
6
Western Blot Analysis of Protein Expression
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8 x 106 cells were harvested and protein extract preparation, quantification was performed as previously described by Gustavsson et al. [17 (link)]. Protein lysate (20 μg) were run on a NuPAGE 10% Bis-Tris gel (Invitrogen) and blotted on to a PVDF membrane using the iBLot® Dry Blotting System (Invitrogen). The membrane was blocked in 5% Milk/PBS before incubating with primary antibodies. Protein expression were assessed using the following antibodies: SOX11 monoclonal antibody [28 (link)], mouse anti GAPDH antibody (G8795, Sigma-Aldrich, St Louis, MO, USA) and EZH2 monoclonal antibody (Clone 11/EZH2, BD Transduction Laboratories, Franklin Lakes, NJ, USA). A HRP-labeled anti mouse antibody (P0260, DAKO, Glostrup, Denmark) was used for detection. Proteins were developed using SuperSignal West Femto Max Sensitivity Substrate (Pierce Biotechnology, Rockford, IL, USA) and images retrieved using a CCD-camera (Odyssey FC Imager from LI-COR Biosciences UK Ltd, Cambridge, England).
7
Comprehensive Antibody Panel for Fibrosis Analysis
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The following antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA): goat anti-COL1A1, anti-p-p70s6k (Thr389), and anti-Akt; mouse anti-α-SMA, anti-LC-3, anti-mTOR, and anti-GNMT; rat anti-CD3; and rabbit anti-p70s6k, anti-p62, anti-p-mTOR (Ser2448), and anti-COL4A2. Mouse anti-p-Akt (Ser473) and rabbit ant-iNOS and anti-MPO antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Rat anti-F4/80 and rabbit anti-ICAM-1 and anti-VCAM-1 antibodies were obtained from Abcam (Cambridge, MA, USA). Both the mouse anti-GAPDH antibody and Masson’s trichrome staining kit were obtained from Sigma–Aldrich (St. Louis, MO, USA). The mouse anti-TGF-β antibody was obtained from R&D Systems (Minneapolis, MN, USA).
8
Alzheimer's Disease Pathways Modulation
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Rabbit-anti-DEK antibody was obtained from Abcam, UK. Anti-Rabbit Cleaved Caspase-3 (Asp175) antibody, anti-Caspase-3 (8G10) antibody, anti-pAKT (ser473) antibody, anti-AKT antibody were obtained from CST, USA. HA-Tag (26D11) mouse antibody was obtained from AbMart. Mouse anti-GAPDH antibody was obtained from Sigma-Aldrich, USA. HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Santa Cruz Biotechnology, USA. The ApopTag® kit were purchased from Millipore, USA. Aβ1-42 peptide (Sigma, USA) was dissolved in sterile distilled water at a concentration of 1 mM and stored at − 20 °C. Cells were transfected with equal amounts of miRNA-138 mimic and/or miRNA-138 inhibitor for 48 h, and then treated with 20 μM Aβ42 for 24 h.
9
Western Blot Analysis of Signaling Proteins
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Proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad, Hercules, California, USA). Then the membrane was blocked with 5% non-fat milk and incubated with rabbit anti-VLDLR antibody (1:400; Abgent), rabbit anti-SP3 antibody (Abgent, San Diego, California, USA) (1:400), mouse anti-GAPDH antibody (1:3000; Sigma-Aldrich), mouse anti-c-Myc antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, California, USA), rabbit anti-p44/42 MAPK (Erk1/2) (1:1000; Cell Signaling Technology [CST], Danvers, Massachusetts, USA), rabbit anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (1:1000; CST), mouse anti-SAPK/JNK (56G8) antibody (1:1000; CST), rabbit anti-phospho-SAPK/JNK (Thr183/Tyr185) (81E11) antibody (1:1000; CST), rabbit anti-p38 MAPK (D13E1) antibody (1:1000; CST), or rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (12F8) antibody (1:1000; CST). The proteins were detected with enhanced chemiluminescence reagents (Thermo Scientific, Waltham, Massachusetts, USA).
10
Quantitative Western Blot Analysis of Pituitary Proteins
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