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15 protocols using gapdh

1

Protein Expression Analysis in Treated Cells

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Cells were treated for up to 3 days for short-term conditions and 6 days for long-term conditions with treatment replenishment on day 4. Total protein lysates were extracted using a Nonidet TMP 40-based lysis buffer (NP-40) and quantified using the Bio-Rad Bradford protein assay. Protein samples were separated via SDS-PAGE (8–12%) in reducing conditions and transferred to nitrocellulose membranes. The latter were blocked in 5% non-fat milk for 1 h and then incubated with primary antibodies against the following antigens at 4 °C overnight: G6PD (Abcam, Waltham, MA, USA) (1:1000), TKT (Cell Signaling, Danvers, MA, USA) (1:1000), Poly (ADP-ribose) polymerase (PARP) (Santa Cruz, Dallas, TX, USA) (1:1000), phosphorylated H2A histone family member X (γH2AX) (Cell Signaling) (1:1000), and GAPDH (Abnova, Taipei, Taiwan) (1:20,000). Membranes were incubated the next day for 1 h against the corresponding secondary antibodies at different optimized dilutions. The immunoreactive bands were visualized using a ClarityTM western ECL substrate (ECL, Bio-Rad) and by the ChemidocTM MP imaging System (Bio-Rad). Densitometric ratios were calculated using ImageJ software for image processing and analysis in Java.
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2

Antibody Sourcing for Western Blotting

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Antibodies were purchased as follows: UCH-L1 (381000) from Thermo Fisher Scientific (Rockford, IL, USA); myc (sc-40) and HSC-70 (sc-7298) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Flotillin-2 (610383) from BD Biosciences (San Jose, CA); Flag (F3165) and β-actin (A1978) from Sigma-Aldrich (St. Louis, MO, USA); and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; H00002597-M3) from Abnova (Taipei, Taiwan). Anti-mouse (NA931V) and anti-rabbit (NA934V) secondary antibodies for Western blotting were purchased from GE Healthcare (Little Chalfont, United Kingdom).
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3

Western Blot Analysis of Cell Proteins

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Cells were washed with PBS and harvested using the RIPA buffer (Beyotime, Nanjing, China). Protein concentration was determined by BCA assay (Beyotime, Nanjing). Total protein (20 µg) was separated using 12% SDS-PAGE, transferred to a nitrocellulose membrane and blocked with 5% milk at room temperature for 1 h. After denaturation, the total protein was separated using SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes (Beyotime, Nanjing). After blocking the proteins with 5% skimmed milk (Beyotime, Nanjing) for 1 h, the PVDF membranes were incubated with rabbit anti-mouse monoclonal antibodies against TIMELESS (1:1000 dilution; cat. no. 109512; Abcam), ESPL1 (1:500 dilution; cat. no. PAB0608; Abnova, Taipei, China) and GAPDH (1:1000 dilution; cat. no. 8245; Abcam, Cambridge, UK) overnight at 4 °C in a shaking incubator. The membranes were then washed with Tris-buffered saline containing Tween-20 and incubated with the respective anti-rabbit IgG secondary antibodies (1:2000 dilution; cat. no. 150077; Abcam) for 1.5 h at room temperature. The immunoblots were visualized using the Odyssey Infrared Imaging System (Li-COR Biosciences, Inc., Lincoln, NE, USA).
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4

RAGE-Mediated Inflammatory Response

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Monocyte chemoattractant protein-1 (MCP-1) was purchased from R&D Systems (MN, USA). Primary antibodies for human RAGE, IkBα, NF-κB p65, phosphorylated NF-κB p65, CCR2, and GAPDH were purchased from Abnova (Taipei, Taiwan). Alexa 488 conjugated secondary antibody, Alexa Fluor 555 Phalloidin, and DAPI were purchased from Beyotime (Shanghai, China). The siRNA duplexes against human RAGE (ID: 110859) were purchased from Ambion Life Technologies (NY, USA). The RAGE shRNA lentiviral particles and control shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (USA). The CCR2 neutralizing antibody and CCR2 Isotype control were purchased from Santa Cruz Biotechnology (USA). The CCR2 antagonist (C28H34F3N5O4S, CAS 445479-97-0) was purchased from Merck Millipore (USA).
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5

Regulation of BMSC Signaling Pathways

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The BMSCs transfected with miR-214 or anti-miR-214 plasmids, or treated with JNK inhibitor or p38 inhibitor were lysed on ice for 30 min in RIPA lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitor. The protein concentration was determined by Bradford assay (Bio-Rad) on a microplate spectrophotometer (Tecan). A total of 40 µg proteins was resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and electro-transferred onto polyvinylidene difluoride (PVDF) membranes (Pall Corporation, New York, NY, USA). The membranes were incubated with the appropriate primary antibodies [anti-fibroblast growth factor (FGF; 1:1,000; 9740), anti-phosphorylated (p-)JNK (1:2,000; 4668), anti-p-p38 (1:2,000; 4511) (all from Cell Signaling Technology, Danvers, MA, USA) and GAPDH (1:5,000; H00002597-D01P; Abnova, Taiwan, China) at 4°C overnight. The membranes were then incubated with an HRP-conjugated secondary antibody (Xi'an Kehao Biological Engineering Co. Ltd, Xi'an, China) and developed by enhanced chemiluminescence (ECL; Millipore, Billerica, MA, USA). Protein expression was analyzed using an Odyssey Two-Color Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
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6

Quantifying Collagen-I and GAPDH in Tissues

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Operated conjunctival tissues were harvested after 28 days and processed as described previously.26 (link) Antibodies against collagen type I α 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Abnova Corporation (Taipei, Taiwan) and Santa Cruz Biotechnology (Santa Cruz, CA) respectively. Horseradish peroxidase–conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Densitometric quantitation was performed using Image Lab software version 6.0.1 (Bio-Rad Laboratories, Hercules, CA). Normalization to correct for variations in loading was performed using GAPDH as the housekeeping protein.
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7

Immunoprecipitation of Tagged Proteins

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Cells were harvested using pre-chilled PBS and lysed using 1% NP-40 lysis buffer with protease inhibitor (Roche). HA-tagged or Myc-tagged fusion proteins were immunoprecipitated by incubation overnight at 4°C with anti-HA or anti-Myc agarose beads (Sigma) and bound protein identified by Western blotting using antibodies against HA (Covance), Myc (BD Biosciences), or CUL1 (Zymed). Antibodies against tubulin (Sigma) or GAPDH (Abnova) were used on the loading controls.
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8

Protein Expression Analysis by Western Blot

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Total protein isolated from cells was heat denatured in Laemmli buffer, resolved by SDS-PAGE and transferred to PVDF membrane. These membranes were then probed for specific proteins using the cognate antibodies for CASEIN KINASE 1α (CK1α) (Santa Cruz Biotechnology), CYCLIN D1 (CCND1) (Cell Signaling), PYGOPUS2 (Abcam), CTNNB1 (Cell Signaling), S45 phospho-CTNNB1 (Cell Signaling), GAPDH (Abnova), β-ACTIN (Santa Cruz Biotechnology) and α-TUBULIN (Sigma). Nuclear extraction was performed using the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce).
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9

Immunoblotting Analysis of Cellular Proteins

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Protein samples were resolved in SDS-PAGE gels, and proteins were subsequently transferred to polyvinylidene difluoride (PVDF) membranes (GE Healthcare). Membranes were blocked with Tris-buffered saline and 0.1% (vol/vol) Tween 20 containing 5% non-fat dry milk, and then probed with the indicated antibodies. Antibodies against FBP1 (611286 from BD Biosciences, Franklin Lakes, NJ; and GTX115154 from GeneTex, San Antonio, TX), eIF4G (GTX115154 from GeneTex), CstF-64 (sc-28201 from Santa Cruz Biotechnology, Santa Cruz, CA), FLAG (F3165 from Sigma, St Louis, MO), His (OB-05 from Calbiochem, LaJolla, CA), HA (H9658 from Sigma), PARP (sc-7150 from Santa Cruz), Lamin A/C (sc-20681 from Santa Cruz), GAPDH (H00002597-M01 from Abnova, Taiwan), and actin (MAB1502 from Millipore, Billerica, MA) were used. EV71 viral proteinase 3C and 3D monoclonal antibodies were generated from recombinant 3Cpro and 3Dpol proteins in our lab. For secondary staining, membranes were washed and incubated with HRP-conjugated anti-mouse antibody or HRP-conjugated anti-rabbit antibody. HRP was detected using the Western Lightning Chemiluminescence Kit (PerkinElmer Life Sciences, Boston, MA).
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10

Western Blotting of Cellular Organelles

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Cell fractionation from whole TAs and protein extraction were performed as described47 (link). Then 25 µg of protein were loaded onto Novex NuPAGE® 4–12% Bis–Tris precast gel (Life Technologies). The membrane was probed with primary monoclonal antibodies directed against histone H3 (Cell Signaling Technology®, 1:10,000), GAPDH (Abnova, 1:15,000), EEA1 (Sigma Aldrich, 1:1,000) or GM-130 (BD Laboratories, 1:500). An incubation with sheep anti-mouse (Jackson Laboratory, 1:15,000) or goat anti-rabbit (Invitrogen, 1:10,000) horseradish peroxidase conjugated secondary antibodies allowed visualisation using Pierce™ ECL Western Blotting Substrate (Thermofisher Scientific).
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