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Pierce 1 step transfer buffer

Manufactured by Thermo Fisher Scientific
Sourced in United States

The Pierce™ 1-Step Transfer Buffer is a pre-formulated buffer designed for efficient transfer of proteins from polyacrylamide gels to membranes during Western blotting procedures. The buffer is ready-to-use and does not require any additional preparation.

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4 protocols using pierce 1 step transfer buffer

1

Protein Expression Analysis by Western Blot

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The total protein from each group was extracted by RIPA lysate, and the concentration of each group was determined by BCA method (Beyotime, China). The SDS-PAGE gel electrophoresis was performed with 30 μg of protein in each group. After electrophoresis, the target protein gel was cut off from the corresponding position referring to the prestained protein. The target protein was transferred to the PVDF membrane by semidry transfer method (Pierce™ 1-Step Transfer Buffer, Thermo, USA) and blocked at room temperature for 1∼2 h and then hybridized with rabbit anti-rat β-actin antibody (1 : 1000, CST, USA), rabbit anti-rat Fyn antibody (1 : 300, Santa Cruz, USA), and rabbit anti-rat STAT5 antibody (1 : 300, Santa Cruz, USA), and then incubated with goat anti-rabbit HRP-IgG antibody after washing for three times (1 : 5000, Bioss, China). The PVDF membranes were fully washed with TBST, and the images were developed by ECL chemiluminescence (ChemiDoc XRS + Imaging System, Bio-Rad, USA). The gray values of each band were read by Image Lab 4.0 software, and β-actin was used as an internal control to analyze the expression levels.
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2

Western Blot Analysis of AP2M1 Protein

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SDS-PAGE gels were transferred to a polyvinylidene fluoride (PVDF) membrane using a Pierce Power Blotter (Thermo Scientific) in Pierce 1-Step Transfer Buffer (Thermo Fisher). PVDF membranes were blocked using EveryBlot Blocking Buffer (Bio-Rad) for 5 minutes at room temperature. The membrane was incubated for 30 minutes with anti-AP2M1 antibody (Abcam: 75995) at 1:1000 in EveryBlot Blocking Buffer (Bio-Rad). Membranes were washed in TBS-T and incubated for 30 minute with IRDye® 800CW goat anti-rabbit IgG secondary antibody (Licor: 926-32211) at 1:20,000 in EveryBlot Blocking Buffer. Blots were imaged using a ChemiDoc MP Imaging System (Bio-Rad) and images were analyzed using BioRad ImageLab software (v.6.1.0 build 7).
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3

Polyacrylamide Gel Electrophoresis and Western Blot

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7.5% polyacrylamide gels were cast and loaded with 50 µg of sample mixed with Laemmli buffer (1:1) that was previously boiled for 5 minutes. Gels were run for 90 minutes at 120 V. Gels were then stained with GelCode (Thermo-Fisher) following manufacturer’s directions. For western blot, 100 µg were loaded into precast 12% SDS-PAGE gels (Biorad) and run at 200 V for 45 minutes. Gels were transferred to a PVDF membrane using a semi-dry transfer system (Thermo-Fisher) with Pierce 1-step transfer buffer (Thermo-Fisher). The membrane was activated with methanol and then blocked overnight at 4 °C with 3% BSA. Primary antibody (keratocan, sc-66941, Santa Cruz) was used at a dilution of 1:200 in 3% BSA and incubated overnight at 4 °C. Three 5-minute washes with TBST were performed under agitation, and then the HRP-linked secondary antibody (A0545, Sigma-Aldrich) at a dilution of 1:1000 was incubated for an hour at room temperature. Another set of washing steps was carried out and then membranes were developed with Western Chemiluminescent HRP Substrate (Fisher Scientific). Membranes were imaged with GelDoc (Biorad).
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4

Hinokitiol Modulates Cell Signaling Pathways

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Hinokitiol (99%, Figure 1A), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), collagenase type IV, and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Type I collagen was purchased from Corning (NY, USA). The β‐actin antibody, Dulbecco's modified Eagle's medium (DMEM), L‐glutamine‐penicillin‐streptomycin, fetal bovine serum, Pierce™ 1‐Step Transfer Buffer, SuperScript™ IV First‐Strand Synthesis System Kit, and Fast SYBR™ Green Master Mix were purchased from Thermo Fisher (Waltham, MA, USA). The NucleoSpin® RNA kit was purchased from Macherey‐Nagel (Düren, Germany). Primary antibodies against Bax, Bcl‐2, phospho‐NF‐κB p65 (Ser536), IκBα, cleaved caspase‐3, and PARP were purchased from Cell Signaling (Beverly, MA, USA). The glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody was purchased from GeneTex (Irvine, CA, USA). Hybond‐P polyvinylidene difluoride (PVDF) membrane, the enhanced chemiluminescence (ECL) western blotting detection reagent, horseradish peroxidase (HRP)‐conjugated donkey anti‐rabbit immunoglobulin G (IgG), and the sheep anti‐mouse IgG were purchased from Amersham (Buckinghamshire, UK). CF488A Donkey anti‐mouse IgG and CF594 Donkey anti‐rabbit IgG were purchased from Biotium (Fremont, CA, USA). Hinokitiol was dissolved in 0.1% dimethyl sulfoxide (DMSO) and stored at 4°C.
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