Total lysates were prepared in PRO-PREP buffer (iNtRON Biotechnology). The preparation of the cell lysates and the western blot analysis were performed as previously described, using the indicated antibodies. 16
Pro prep buffer
Manufactured by iNtRON Biotechnology
Sourced in United States
PRO-PREP buffer is a protein extraction reagent designed for efficient extraction of proteins from various biological samples. It is a balanced buffer solution that helps to solubilize and stabilize proteins, making them suitable for further analysis and applications.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase, by Santa Cruz Biotechnology (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (4 mentions)
Chemidoc xrs imaging system, by Bio-Rad (3 mentions)
Microchemi 4.2 system, by DNR Bio-Imaging Systems (3 mentions)
Vimentin, by Thermo Fisher Scientific (2 mentions)
E cadherin 24e10, by Cell Signaling Technology (2 mentions)
Pgc 1α, by Abcam (2 mentions)
Vimentin, by Santa Cruz Biotechnology (2 mentions)
Phospho erk, by BD (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
E cadherin, by BD (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Shmt2, by Cell Signaling Technology (1 mentions)
Igfbp5, by Santa Cruz Biotechnology (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
26 protocols using pro prep buffer
1
Cell Lysis and Western Blotting
2
RAW 264.7 Osteoclastogenesis Assay
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RAW 264.7 cells seeded at a density of 8 × 103 cells/well in 6-well plates were cultured with DMEM for 24 h at 37°C in a 5% CO2 incubator. After 24 h, the medium was replaced with α-MEM containing 50 ng/mL RANKL and the sample extract. After 3 days, the cells were lysed with PRO-PREP buffer (iNtRON Biotechnology, Seongnam, Korea) to extract the expressed protein. Nuclear protein was extracted with NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific). The concentration of the protein extract was determined using a protein assay with bovine serum albumin (BSA) as the standard. Proteins were resolved via SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked in 5% BSA and incubated overnight at 4°C with the primary antibodies specific for the targeted molecules. The membranes were then incubated with the corresponding secondary antibodies, and the targeted proteins in the membrane were visualized by enhanced chemiluminescence (ECL) reagents. The relative intensities of the bands were visualized and analyzed using the ChemiDoc XRS+ imaging system (BIO-RAD Laboratories, Hercules, CA, USA) equipped with Image Lab software.
3
Western Blot Analysis of EMT Markers
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To prepare whole-cell extract, cells were lysed using Pro-prep buffer (Intron Biotechnology, Seoul, Korea) including protease inhibitors. Similar amounts of protein extracts were resolved via SDS-PAGE and transferred to polyvinylidene fluoride membranes. The membranes were probed with primary antibodies followed by incubation with secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, CA, USA). β-actin was used as a loading control in western blot analysis. Antibodies used for this study were as follows: E-cadherin (24E10) (#3195, Cell Signaling Technology, Danvers, USA), vimentin (#MA5-11883, Thermo Fisher Scientific, MA, USA), and β-actin (#3700, Cell Signaling Technology).
4
CCL7 Regulation of CCR3 Expression
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Example 3
In order to determine the effect of CCL7 on CCR3 expression, HCT116 colorectal cancer cells were stably transfected with GFP (control) or CCL7. CCR1, CCR2, CCR3, CCR5, and CCL7 levels were determined by Western blot and FACS analyses.
For the Western blot, lysates from transfected HCT116 cells were obtained using Pro-prep buffer (Intron Biotechnology, Seoul, Korea), including protease inhibitors. 20-60 μg the protein extract was resolved by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes and probed with antibodies against CCR1, CCR2, CCR3, CCR5, CCL7, and β-actin, as a loading control. Secondary antibodies conjugated to horseradish peroxidase were used to image the membrane.
For the flow cytometry, HCT116 colorectal cancer cells were incubated with 200 ng/mL recombinant CCL7 for up to 12 hours. Following incubation, the cells were washed, blocked, and then stained with anti-human antibodies against CCR1, CCR2, CCR3, or CCR5 followed by staining with a PE-conjugated secondary antibody.
Overexpression of CCL7 in colorectal cancer cells significantly and dramatically increased protein expression of CCR3 compared with GFP controls (
5
Whole-Cell Extract Protein Analysis
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To prepare whole-cell extracts, the cells were lysed using Pro-Prep buffer (Intron Biotechnology, Seoul, South Korea) containing phosphatase and protease inhibitors. Equal amounts of lysate were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking with 5% skim milk, the membranes were incubated with the indicated primary antibodies, washed, and incubated with the appropriate secondary antibodies. The following antibodies were used: SHMT2 (Cell Signaling Technology, Danvers, MA, no. 12762), IGFBP5 (Santa Cruz Biotechnology, sc-515116), vimentin (Thermo Fisher Scientific, Waltham, MA, no. MA5-11883), E-cadherin (24E10) (Cell Signaling Technology, no. 3195), and β-actin (Cell Signaling Technology, no. 3700). β-actin was used as the loading control for western blotting. Protein bands were detected using an enhanced chemiluminescence reagent.
6
Cartilage Protein and RNA Extraction
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Healthy cartilage devoid of any damage in the femoral condyles and tibial plateau joint, and degenerative cartilage showing clear damage, was collected from the knee of an OA patient. Almost 50 mg of cartilage frozen in liquid nitrogen was pulverized using a mortar and pestle. Total protein was extracted using PRO-PREP buffer (iNtRON Biotechnology, Seongnam-Si, Korea), according to the manufacturer's instructions. Total RNA was extracted using the TRIzol reagent (Invitrogen Life Technologies Inc., Carlsbad, CA, USA).
7
Western Blot Analysis of Neuronal Markers
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SH-SY5Y cells or brain tissues (n = 7 per group) were lysed in PRO-PREP™ buffer (iNtRON Biotechnology, Seongnam, Korea) to obtain proteins. The protein amount from each sample was calculated using the Bio-Rad Bradford kit (Bio-Rad). An equal amount (30 μg) of protein was loaded and separated using 10–12% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Millipore Corp., MA, USA). Blocking was performed with 5% skimmed milk for 2 h, and the blots were incubated for 1 d with primary antibodies against Bax, Bcl-2, cleaved-caspase-3, BDNF, CREB, pCREB, ERK, pERK, AKT, pAKT, GSK3β, pGSK3β, and GAPDH (dilution 1 : 1000). The blots were then incubated with secondary antibodies (dilution, 1 : 2000). Protein bands were analyzed using the Chemi DocXRS+imaging system (Bio-Rad).
8
Peroxiredoxin Expression Analysis in Tissues
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Lysates of tissue were prepared in ice-cold PRO-PREP buffer (iNtRON Biotechnology Inc., Korea). Proteins were separated on 12% SDS-polyacrylamide gels and then transferred to NitroBind nitrocellulose (Osmonics Inc., USA). The membrane was blocked by blocking buffer and incubated with the following primary antibodies (diluted 1:3000): anti-peroxiredoxin 1, anti-peroxiredoxin 2 and anti-peroxiredoxin 3, anti-peroxiredoxin-SO2/3 (Abfrontier, Korea). Following incubation, membranes were washed and incubated with anti-goat, anti-rabbit, and anti-mouse IgG horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000, Thermo, Scientific, Korea) for 1 h at room temperature. After the removal of excess antibodies by washing, specific binding was detected using an ECL kit (Abfrontier). Anti-β-actin antibody (1:5000, Santa Cruz) was used as a loading control. Band intensities were analyzed using Multi Gauge version 3.0 software (Fuji).
9
Western Blot Analysis of Sulf-1 and Sulf-2
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Total lysates were prepared in PRO-PREP buffer (iNtRON Biotechnology, Seoul, Korea). Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin as a standard. Twenty micrograms of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes. Membranes were soaked in 5% non-fat dried milk in TBST (10 mmol/L Tris/HCl pH 7.5, 150 mmol NaCl, and 0.05% Tween-20) for 1 hour, followed by incubation for 16 to 18 hours with primary antibodies against Sulf-1, Sulf-2, and β-actin at 4°C. Membranes were then washed three times with TBST for 10 minutes, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. Finally, membranes were rinsed three times with TBST for 10 minutes, and antigen-antibody complexes were detected using an enhanced chemiluminescence detection system (LAS-3000; Fujifilm, Tokyo, Japan).
10
Western Blot Analysis of RANKL-induced Osteoclastogenesis
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RAW 264.7 cells (8 × 103 cells/well) were seeded in 6-well plates with DMEM culture medium supplemented 10% FBS and 1% penicillin-streptomycin and incubated for 24 h at 37 °C in an atmosphere of 5% CO2. After incubation, the cells were treated by 50 ng/mL RANKL and extracts with α-MEM (containing 10% FBS and 1% penicillin-streptomycin). After 3 days, the whole-cell extract was collected by the addition of PRO-PREP buffer (iNtRON Biotechnology). The nuclear protein was collected by using NE-PER nuclear and cytoplasmic extraction reagents according to the manufacturer’s instructions (Thermo Scientific). A BSA protein assay was used to determine the protein concentration of all the extracts. The extracts were subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were blocked with 5% BSA, probed with primary antibodies and incubated at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibodies were then added. The targeted proteins were visualized by using enhanced chemiluminescence. The images were obtained by using the BIO-RAD ChemiDoc XRS+ (BIO-RAD, Philadelphia, PA, USA) imaging system and Image Lab software.
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