Total protein was extracted by lysing cells in radio-immunoprecipitation assay (RIPA) lysis buffer (GenDEPOT, Barker, TX, USA) containing a protease inhibitor cocktail (GenDEPOT) for 30 min on ice. The protein concentration was determined using a bicinchoninic acid protein assay (Sigma-Aldrich). Equal amounts of protein were loaded onto a 12% (v/v) sodium dodecyl sulfate-polyacrylamide gel for electrophoresis. The separated proteins were blotted onto a polyvinylidene fluoride membrane (Amersham Biosciences, Piscataway, NJ, USA). Membranes were first blocked with 3% BSA in Tris-buffered saline with 0.1% Tween-20 at RT for 1 h and then incubated overnight at 4 °C with primary polyclonal antibodies (1:1000 dilution; Table 2 ). The blots were then incubated with peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies (7076 and 7074, respectively, Cell Signaling Technology, Danvers, MA, USA) diluted at 1:10,000 for 1 h at RT. The target proteins were visualized using an enhanced chemiluminescence kit (Amersham Biosciences), imaged using an Amersham Imager 680 (Amersham Biosciences) and quantified with ImageJ software. The density of each band was normalized to that of β-actin.
Ripa lysis buffer
Manufactured by GenDEPOT
Sourced in United States
RIPA lysis buffer is a commonly used detergent-based cell lysis and extraction buffer. It is designed to solubilize and extract proteins from cells and tissues for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by GenDEPOT (2 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Image lab 3, by Bio-Rad (2 mentions)
Dyne ecl star western blotting detection kit, by Dyne Bio (2 mentions)
Polyvinylidene fluoride membrane, by Cytiva (1 mentions)
Enhanced chemiluminescence kit, by Cytiva (1 mentions)
Bicinchoninic acid protein assay, by Merck Group (1 mentions)
Amersham imager 680, by Cytiva (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Las 4000 mini system, by GE Healthcare (1 mentions)
Las 4000, by Cytiva (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Tissuelyzer 2, by Qiagen (1 mentions)
Hrp conjugated anti mouse antibody, by Jackson ImmunoResearch (1 mentions)
Nc membrane, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Ecl plus kit, by Merck Group (1 mentions)
11 protocols using ripa lysis buffer
1
Western Blot Protein Quantification
2
Spinal Cord Injury Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Only the injured spinal cord was collected, frozen with liquid nitrogen, and smashed into small pieces with a hammer. After 30 min of incubation with RIPA lysis buffer (Gen Depot, Grand Island, NY, USA) and proteinase inhibitor solution (Gen Depot), centrifugation was performed for 15 min at 12,000× g at 4 °C and the upper layer was separated. The Bradford assay was used to determine the protein concentration in the supernatant. A protein of 20 μg was separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked for 1 h with 5% skim milk and incubated overnight with primary antibodies. The following primary antibodies were used: nestin (neural progenitor stem cells, sc-23927), glial fibrillary acidic protein (GFAP, astrocytes, sc-33673), and β-III-tubulin (immature neurons, sc-80005). Afterwards, the membrane was incubated with anti-mouse secondary antibody (sc-516102) for 1 h, and the protein band was visualized using enhanced chemiluminescent substrates (ECL) (Bio-Rad, Hercules, CA, USA) and quantified using an LAS 4000 mini system (GE Healthcare, Lafayette, CO, USA).
3
Western Blot Analysis of Macrophage Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Macrophages were harvested and resuspended in RIPA lysis buffer (GenDEPOT, Baker, TX, USA). Concentrations of proteins were determined using a BCA protein assay (ThermoScientific, Rockford, IL, USA). Proteins (30 μg) were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. Membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and 5% skim milk, incubated with specific primary antibodies recognizing β-actin, COX-2, iNOS, and arginase-1, and then incubated HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Signals were developed using an enhanced chemiluminescence system (Pierce Biotechnology Inc., Rockford, IL) [16] (link).
4
RNA and Protein Extraction from Mouse Embryos
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole embryos from C57BL/6 mice were homogenized in TRIzol reagent (Ambion, Austin, TX, USA) for extraction of total RNA or RIPA lysis buffer (GenDEPOT, Barker, TX, USA) for extraction of total protein using a TissueLyzer II (Qiagen, Hilden, Germany) according to the manufacturer’s manual.
5
Macrophage Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Macrophages were harvested and resuspended in RIPA lysis buffer (GenDEPOT, Baker, TX, USA). Concentrations of proteins were determined using a BCA protein assay (ThermoScientific, Rockford, IL, USA). Proteins (30 μg) were resolved by 10% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. Membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and 5% skim milk, incubated with specific primary antibodies recognizing β-actin, COX-2, iNOS, arginase-1, caspase-1, and caspase-11, and then incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Signals were developed using an enhanced chemiluminescence system (Pierce Biotechnology Inc., Rockford, IL, USA) [14 (link)].
6
Western Blot Quantification of Liver Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins extracted from whole liver tissue were obtained using RIPA lysis buffer (GenDEPOT, Hanam, Republic of Korea). Twenty micrograms of these proteins was then loaded onto a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel. Following electrophoresis, the proteins were transferred to a nitrocellulose (NC) membrane (pore size 0.45 μm, Bio-Rad, Hercules, CA, USA). The transferred membrane was incubated with 1X EzBlock Chemi solution (ATTO, Tokyo, Japan) for 30 min, followed by overnight incubation with primary antibodies at 4 °C. The primary antibodies used are listed in Table 2 . Subsequently, the membrane was incubated with a secondary HRP-conjugated anti-mouse antibody (1:5000, Jackson Immunoresearch, West Grove, PA, USA) at room temperature for 1 h. β-Actin served as the protein loading control. The transferred membranes were visualized using the Dyne ECL STAR Western blotting detection kit (Dyne Bio, Seongnam, Republic of Korea), and the results were quantified using an image analyzer (Image Lab 3.0, Bio-Rad, Hercules, CA, USA). All of the primary antibodies are described in Table 2 .
7
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with DPBS, lysed with RIPA lysis buffer (GenDepot, Barker, TX) containing 1% of 100x protease inhibitor cocktail (GenDepot, Barker, TX), and incubated for 30 min on ice. Lysates were centrifuged at 19,000 g for 30 min at 4°C, and the supernatant was mixed with 25% of 4x denaturating buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, and 20% glycerol with bromophenol blue) and heated for 5 min. The proteins were separated through 10% SDS-PAGE gels and were transferred to a nitrocellulose membrane by Mini Trans-Blot Cell (Bio-Rad, CA). The membrane was blocked in 5% BSA in TBS containing 0.1% Tween 20 (TBS-T) for 1 h and incubated overnight with the intended antibodies in and 3% BSA. Excess primary antibodies were then removed by washing with TBS-T for 3 times. The membrane was then incubated with HRP-conjugated secondary antibodies (0.1 μg/mL, anti-rabbit) for 1 h. After three washes with TBS-T, bands were visualized by western blot and exposed to X-ray film, or by ChemiDoc Imaging Systems (Bio-Rad). The original film images were supported in Figs.
8
Western Blot Analysis of COX and PGES
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein expression was assessed with western blots. Briefly, collected cells were lysed with RIPA lysis buffer (GenDEPOT, Houston, TX, USA), and protein concentration was quantified using the BCA assay. Protein samples were then resolved by SDS-PAGE and transferred onto a nitrocellulose membrane (GE Healthcare). After blocking, the membranes were incubated with the respective primary mouse antibodies: anti-cyclooxygenase-1 (COX-1; 1:1000), anti-cyclooxygenase-2 (COX-2; 1:500; Abcam, Cambridge, UK), anti-microsomal PGE synthase-1 (mPGES-1; 1:1000), anti-microsomal PGE synthase-2 (mPGES-2; 1:500), anti- cytosolic PGE synthase (cPGES; 1:200; Cayman Chemicals), or anti-β-actin (1:1000; Cell Signalling Technology, Beverly, MA, USA). The membranes were then incubated with peroxidase-conjugated affinity-purified goat anti-rabbit IgG or goat anti-mouse IgG secondary antibody (1:10000; GenDEPOT), and the protein bands were detected using an ECL Plus kit (Millipore Corporation, Billerica, MA, USA).
9
Western Blot Analysis of HSV-1 Thymidine Kinase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For western blotting, whole cell lysates were prepared using RIPA lysis buffer (GenDEPOT, Katy, TX, USA). Lysates were then centrifuged at 14,000× g for 10 min at 4 °C. Supernatants were loaded to SDS-PAGE (4–15%) gradient gels and transferred to PVDF membranes (Millipore, USA cat. no. IPVH00010). Membranes were incubated overnight at 4 °C with primary antibody, herpes simplex virus type 1 thymidine kinase (SC-28037, Santa Cruz, Dallas, TX, USA), and washed three times in PBS with 0.1% Tween 20 prior to 1 h incubation with secondary antibody, anti-Goat IgG (no. A50-101P, Bethyl, Montgomery, TX, USA), at room temperature. Proteins were then detected using chemi-doc system (no. CAS400SM, Davinch-K, Gangnam-gu, Seoul, Korea).
10
Western Blot Analysis of Liver Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver tissue proteins were extracted using RIPA lysis buffer (GenDEPOT, Hanam, Republic of Korea). The extracted proteins (11 μg) were then loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis. The separated proteins were transferred onto nitrocellulose (NC) membranes (0.45 μm-pore size; Bio-Rad, Hercules, CA, USA), blocked with 1× EzBlock Chemi solution (ATTO, Tokyo, Japan) for 30 min, and incubated with the primary antibodies, α-SMA (1:1000; ab5694, Abcam, Cambridge, UK) and GAPDH (1:1000; GTX100118, Genetex, CA, USA), overnight at 4 °C. Membranes were then incubated with a secondary HRP-conjugated anti-rabbit antibody (1:5000; SA200-500, Jackson Immunoresearch, West Grove, PA, USA) for 1 h at 25 ± 2 °C. GAPDH was used as a protein loading control. Positive protein bands were visualized using the Dyne ECL STAR Western blotting Detection Kit (Dyne Bio, Seongnam, Republic of Korea), and the results were quantified using an image analyzer (Image Lab 3.0, Bio-Rad, Hercules, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!