Hippocampi from KA-treated (1, 3 and 7 days) and control mice were dissected and homogenized in lysis buffer (PRO-PREP reagent, Intron Biotechnology, Sungnam, Rep. of Korea). Cultured primary microglia, BV-2, and HT22 cells were collected by scraping, and the pellet was solubilized in lysis buffer (1X RIPA buffer, #9806, Cell Signaling). After centrifugation, protein concentrations were determined in supernatants using MicroBCA protein assay kits; bovine serum albumin was used as standard (Pierce Chemical, USA). Aliquots containing 30 μg protein were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by enhanced chemiluminescence, according to the manufacturer’s instruction (Amersham).
Pro prep reagent
Manufactured by iNtRON Biotechnology
PRO-PREP is a reagent used for protein extraction and purification. It is designed to efficiently lyse cells and solubilize proteins, facilitating their extraction and isolation from various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal ecl kit, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Cytiva (2 mentions)
Ez capture mg, by ATTO Corporation (2 mentions)
Biomax light 1 film, by Kodak (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Goat anti mouse igg h l hrp, by GenDEPOT (1 mentions)
Anti cd34 antibody, by Abcam (1 mentions)
Goat anti rabbit igg hrp, by GenDEPOT (1 mentions)
Anti α smooth muscle actin antibody, by Abcam (1 mentions)
Immun blot pvdf membrane, by Bio-Rad (1 mentions)
β actin, by Abcam (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
P ikkα β, by Santa Cruz Biotechnology (1 mentions)
10 protocols using pro prep reagent
1
Protein Expression Analysis in KA-Treated Mice
2
Western Blot Analysis of Capsular Proteins
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Capsular tissues were solubilized by sonication in lysis buffer using PRO-PREP reagent (Intron Biotechnology, Daejeon, Republic of Korea), and the concentration of protein was measured using a BCA Protein Assay kit (Thermo-Fisher, Seoul, Republic of Korea). After being denatured by boiling, the protein sample (10 μg for each lane) was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, Boston, MA). The blot was probed with a primary antibody [rabbit polyclonal to CTGF (C Terminus, IHC-plus™) (1 : 100)], mouse monoclonal to TGF-β (1 : 50), goat MPO antibody (1 : 1,000), rabbit polyclonal VEGF (1 : 1,000), or mouse monoclonal β-actin, (1 : 1,000)] in a blocking solution of 5% BSA in Tris-buffered saline containing Tween-20 (5% BSA-TBST) overnight at 4°C and then incubated with peroxidase-conjugated secondary antibodies (1 : 5,000) for 1 h at room temperature. The immunolabeled proteins were detected by chemiluminescence using a SuperSignal ECL kit (Pierce Chemical, Rockford, Ill) and ImageQuant LAS 4000 (GE Healthcare Life Science, Marlborough, MA, USA).
3
Protein Extraction and Immunoblotting Protocol
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Proteins were extracted from cells after three passages with PRO-PREP reagent (Intron Biotechnology, Gyeonggi, Korea), incubated on ice, and centrifuged at 13,000 rpm at 4 °C for 5 min. The supernatant was then transferred to fresh tubes on ice. The protein concentration was analyzed with a Protein Assay Kit (Bio-Rad, Hercules, CA, USA).
The proteins in samples containing 30 mg of protein were separated sequentially on 6% and 2% SDS/PAGE gels and transferred to a polyvinylidene fluoride membrane (Immun-Blot® PVDF Membrane, Bio-Rad, Hercules, CA, USA). Blotted membranes were blocked for 60 min with 3% BSA/5% skim milk and washed. The membrane was incubated overnight at 4 °C with each primary antibodies and 70 min at room temperature with each secondary antibodies.
We employed α-smooth muscle actin as a marker for smooth muscle cells, von Willebrand factor and CD34 as markers for endothelial cells, and β-actin as a housekeeping protein. The following primary antibodies were acquired from Abcam: β-actin (mouse, 1:1,000, #8224), anti-CD34 antibody (rabbit, 1:1,000, #81289), anti-α-smooth muscle actin antibody (rabbit, 1:1,000, #5694), and anti-von Willebrand Factor antibody (rabbit, 1:500, #6994). The secondary antibodies were acquired from GEN Depot and were: goat anti-Mouse IgG (H+L)-HRP (1:10,000, #SA001-500) and goat anti-rabbit IgG-HRP (1:10,000, #SA002-500).
The proteins in samples containing 30 mg of protein were separated sequentially on 6% and 2% SDS/PAGE gels and transferred to a polyvinylidene fluoride membrane (Immun-Blot® PVDF Membrane, Bio-Rad, Hercules, CA, USA). Blotted membranes were blocked for 60 min with 3% BSA/5% skim milk and washed. The membrane was incubated overnight at 4 °C with each primary antibodies and 70 min at room temperature with each secondary antibodies.
We employed α-smooth muscle actin as a marker for smooth muscle cells, von Willebrand factor and CD34 as markers for endothelial cells, and β-actin as a housekeeping protein. The following primary antibodies were acquired from Abcam: β-actin (mouse, 1:1,000, #8224), anti-CD34 antibody (rabbit, 1:1,000, #81289), anti-α-smooth muscle actin antibody (rabbit, 1:1,000, #5694), and anti-von Willebrand Factor antibody (rabbit, 1:500, #6994). The secondary antibodies were acquired from GEN Depot and were: goat anti-Mouse IgG (H+L)-HRP (1:10,000, #SA001-500) and goat anti-rabbit IgG-HRP (1:10,000, #SA002-500).
4
Immunoblotting Analysis of Protein Expression
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The protein cell lysates were isolated by Pro-Prep™ reagent (iNtRON Biotechnology, Seongnam, Korea). Total proteins were separated by electrophoresis on gradient SDS-PAGE gels (8% to 15%) and transferred onto a PVDF membrane (Amersham Biosciences, Bukres, UK). Antibodies against GAPDH, NF-κB, NDRG2, p-IKKα/β, Lamin A/C, and α-actinin were purchased from Santa Cruz Biotechnology. Anti-human PD-L1, p-STAT3, STAT3, α-tubulin, p-IκBα, IκBα, p-NF-κB (p-p65), and PD-1 antibodies were purchased from Cell Signaling (Beverly, MA, USA). Anti-mouse PD-L1 antibody was purchased from R&D Systems (Minneapolis, MN, USA). The blots were visualized by Ez-Capture MG (ATTO Corporation, Tokyo, Japan).
5
Whole Cell Lysate Preparation and Protein Analysis
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For the preparation of whole cell lysates, the cells were lysed on ice in the PRO-PREP™ reagent (iNtRON Biotechnology, Korea) for 20 min. Nuclear and cytoplasmic extracts were prepared using the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, USA), according to the manufacturer’s instructions. The proteins were separated in an SDS-polyacrylamide gel and transferred to a PVDF membrane (Amersham Biosciences, UK), which was then blocked with Tris-buffered saline plus 0.05% Tween-20 (TBST) containing 5% skim milk. The membranes were incubated with specific antibodies overnight at 4°C and washed with TBST. The antibody recognizing COX-2 was purchased from Cayman Chemical (USA), and the antibodies against NDRG2, p-IKKα/β (Ser176), IKKβ, NF-κB p65, Akt1/2, Lamin A/C, and actin were purchased from Santa Cruz Biotechnology. The antibodies for p-IκBα (Ser32/36), IκBα, p-Erk1/2 (Thr202/Tyr204), Erk1/2, and p-Akt (Ser473) were purchased from Cell Signaling Technology Inc. (USA). After incubating the membrane with the appropriate secondary antibodies coupled to horseradish peroxidase followed by enhanced chemiluminescence, the blots were visualized using the Ez-Capture MG (ATTO Corporation, Japan).
6
Western Blot Analysis of LPS-Stimulated Astrocytes
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For western blot, the cells were incubated for 24 h prior to 4 h serum-starvation and stimulated with LPS (100 ng/mL) for the indicated times. Cultured astrocytes were collected by scraping, and the pellet was solubilized in lysis buffer using PRO-PREP reagent (Intron Biotechnology, Sungnam, Korea) with a protease inhibitor cocktail (Sigma P5726). Following normalization of protein content in each sample, 30 µg of the total cellular fraction of each sample was separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transblotted onto nitrocellulose membranes. The blot was probed with primary antibodies, e.g. GDF15, P-p65 (Cell Signaling, #3033S), IκB-α (Santa Cruz, #sc-371), beta-actin (T308, #2965, Cell Signaling, CA, USA) in blocking solution. Membranes were washed for 3 times for 10 min in TBST, and incubated for 1 h with peroxidase labeled secondary antibody (Vector) diluted 1:2000 in TBST. After three further washes, immunolabeled proteins were detected by chemiluminescence using a Supersignal ECL kit (Pierce Chemical) and Biomax Light-1 films (Kodak,USA).
7
Inflammatory Signaling Pathway Analysis
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Hippocampi from LPS-treated (1 and 3 days) control mice and TLR4 knockout mice were dissected and homogenized in lysis buffer. After centrifugation, protein concentrations were determined in supernatants using Micro BCA Protein Assay kits; bovine serum albumin was used as a standard (Pierce Chemical, Rockford, IL, USA). Cultured HT22 cells were collected by scraping, and the pellet was solubilized in lysis buffer using PRO-PREP reagent (Intron Biotechnology, Sungnam, Korea) with a protease inhibitor cocktail. Following normalization of protein content in each sample, 30 μg of the total cellular fraction of each sample was separated by 10 or 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transblotted onto nitrocellulose membranes. The blot was probed with primary antibodies: P-p65 (1:1000, #3033, Cell Signaling), IκBα (1:300, #sc-371, Santa Cruz), Cyclooxygenase 2 (Cox2, 1:500, #sc-166,475, Santa Cruz), inducible nitric oxide synthase (iNOS, 1:500, #n32030, Transduction Laboratories), and β-actin (1:500, #2965, Cell Signaling). The immune complexes were identified using an enhanced chemiluminescence (ECL) detection system (Amersham).
8
Western Blot Protein Expression Analysis
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The plate was washed once with PBS, scraped with a cell scraper, and centrifuged at 4 • C and 13,000 rpm for 3 min to remove suspended solids. Then, a pro-prep reagent (iNtRON Biotechnology, Gyeonggi-do, Korea) was added. Protein quantification was performed using the PRO-MEASURE kit (iNtRON Biotechnology, Gyeonggi-do, Korea). Equal amounts of protein were separated by 10% SDS-polyacrylamide gel electrophoresis and followed by electrophoretic transfer (Bio-Rad Laboratories, Hercules, CA, USA) onto a polyvinylidene difluoride membrane (Merck Millipore, Bedford, MA, USA). The transferred membrane was blocked in 5% skim milk diluted in Tris-buffered saline solution (TBST). After blocking, the slides were washed with TBST three times for 10 min, and the primary antibody was dissolved in 5% bovine serum albumin overnight at 4 • C. After washing the membrane, a horseradish peroxidase-conjugated secondary antibody (Promega, Madison, WI, USA) was reacted at room temperature for 1 h. The membrane was exposed to an enhanced chemiluminescence solution (iNtRON Biotechnology, Gyeonggi-do, Korea). Expression was confirmed using ImageQuant LAS 500 (GE Healthcare Life Sciences, Uppsala, Sweden).
9
Western Blot Protein Detection Protocol
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To extract the total protein, cells were lysed using a Pro-Prep reagent (iNtRON Biotechnology, Seongnam, Korea). Each sample contained the same amounts of protein, as determined by quantifying the protein contents in the lysates. The samples were loaded and separated via SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred onto polyvinylidene difluoride (PVDF, Millipore, Temecula, CA, USA) membranes using the wet transfer method. The membranes were blocked using 5% skim milk for 1 h; then, they were incubated overnight with primary antibodies at 4 °C. After incubation with the primary antibodies, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h (Millipore, Temecula, CA, USA). The signals were detected using chemiluminescence reagents (AbClon, Seoul, Korea) and quantified using ImageJ software.
10
Western Blot Analysis of Astrocyte Signaling
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For western blot, cell scrappers were used to collect cultured astrocytes PRO-PREP reagent (Intron Biotechnology, Sungnam, Korea) with a protease inhibitor cocktail (Sigma P5726, location?) was used as the lysis buffer in order to solubilize the collected cell pellet. Protein content was normalized for 30 µg of the total cellular fraction. Each sample was then separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transblotted onto nitrocellulose membranes. The blot was probed with primary antibodies, p-CTMP, p-AKT (T308), CTMP, p-CREB, and β-actin antibodies (Sigma, USA) in blocking solution. Membranes were washed in TBST (3 times, 10 min each). Peroxidase (1:2000 dilution) (Vector, location?) was used for secondary antibody staining. Samples were washed 3 times then detected for immunolabeled proteins by chemiluminescence using a Supersignal ECL kit (Pierce Chemical, Rockford, IL) and Biomax Light-1 films (Kodak, USA).
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