Gene expression was analyzed using western blot analysis. Cell pellets were lysed on ice in RIPA buffer (Pierce, Rockford, IL) supplemented with Complete Mini EDTA-free Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and Phosphatase Inhibitor Cocktail (Sigma, St. Louis, MO). Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA). Protein (40 μg) was diluted 1:5 in 5X protein loading buffer (Fermentas, Glen Burnie, MD), boiled at 80 °C for 5 min, electrophoresed on a 4–20% Tris-Glycine gel, and transferred using a Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA). Membranes were blocked in 5% Non-fat milk powder (BioRad), incubated with primary antibody overnight at 4 °C, incubated with HRP-coupled secondary antibody 1 h at room temperature, developed with Visualizer Western Blot Detection Kit (Millipore, Billerica, MA), and visualized on a LAS-4000 imager (Fujifilm, Edison, NJ). The following antibodies were used at 1: 1000 dilutions: human anti-CD44 (#3570S, Cell Signaling Technology) and mouse anti-actin (MAB 1501R, Millipore). Secondary antibody, anti-mouse-HRP (Santa Cruz Biotechnology, Santa Cruz, CA) was used at 1:10,000 dilutions.
Protein loading buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Protein Loading Buffer is a laboratory reagent used to prepare protein samples for analysis by gel electrophoresis. It is designed to ensure the proper denaturation, reduction, and solubilization of proteins prior to loading them onto a gel.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Visualizer western blot detection kit, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Las 4000 imager, by Fujifilm (2 mentions)
Complete mini edta free protease inhibitor cocktail, by Roche (2 mentions)
Nonfat milk powder, by Bio-Rad (2 mentions)
Sc30435, by Santa Cruz Biotechnology (2 mentions)
Sc 126, by Santa Cruz Biotechnology (2 mentions)
Sc 7351, by Santa Cruz Biotechnology (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Sc 789, by Santa Cruz Biotechnology (2 mentions)
Semi dry electroblotting, by Bio-Rad (2 mentions)
Sc 1050, by Santa Cruz Biotechnology (2 mentions)
Sc 200, by Santa Cruz Biotechnology (2 mentions)
E6 sc 460, by Santa Cruz Biotechnology (2 mentions)
Sc 188 c 19, by Santa Cruz Biotechnology (2 mentions)
Sc 520, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay, by Merck Group (2 mentions)
11 protocols using protein loading buffer
1
Western Blot Analysis of CD44 Protein
2
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell pellets were lysed on ice in RIPA buffer (Pierce, Rockford, IL) supplemented with Complete Mini EDTA-free Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and Phosphatase Inhibitor Cocktail (Sigma, St. Louis, MO). Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA). Protein(50ug) was diluted 1:5 in 5X protein loading buffer (Fermentas, Glen Burnie, MD), boiled at 80°C for 5 minutes, electrophoresed on a 4-20% Tris-Glycine gel, and transferred using a Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA). Membranes were blocked in 5% Non-fat milk powder (BioRad), incubated with primary antibody overnight at 4°C, incubated with HRP-coupled secondary antibody 1 hour at room temperature, developed with Visualizer Western Blot Detection Kit (Millipore, Billerica, MA), and visualized on a LAS-4000 imager (Fujifilm, Edison, NJ). The following antibodies were used at 1:1000 dilutions: Rabbit anti-FOXM1 (#5436), PLK1(#4513), pCDC2(#9111), CDC2 (#9112), MRE11, Survivin (#4895), mouse anti-STAT3(#9139), pSTAT3 (#9138), ß-Actin (#3700) (Cell Signaling Technology., MA); mouse-anti -pγH2AX (#05636, Millipore., MA), rabbit-anti RAD51 (sc-8348, Santacruz., CA), mouse anti-53BP1 (#612522, BD Transduction Laboratories., CA). Secondary antibodies, goat anti-rabbit-HRP, goat anti-mouse-HRP (Santa Cruz, CA) were used at 1: 10,000 dilution.
3
Western Blot Analysis of YF Vaccine
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed WB analysis on the obtained inactivated YF vaccine (Vero cells) original liquid. The complex conformation of proteins in the original liquid was destroyed in 4 × protein loading buffer (Invitrogen, Carlsbad, CA, USA) at 95 °C for 10 min. The protein was redistributed using 12% SDS-polyacrylamide gel electrophoresis (Solarbio, Beijing, China) and transferred to the PVDF membrane (Millpore, Billerica, MA, USA). The protein was immobilized on the surface of the solid phase and blocked with 5% skimmed milk powder (BD, New York, NY, USA) at room temperature for 1 h. Antibodies against YFV Envelope (E) protein (1:2000, GeneTex, Alton Pkwy Irvine, CA, USA) were incubated with the membrane at 4 °C for 16–24 h. The second antibody IgG labeled with HRP (1:8000, Cytiva, North Logan, UT, USA) was used to bind the first antibody and mixed with an ECL luminescent solution (Cytiva, USA). Finally, the chemiluminescence instrument (Amersham ImageQuant 800, Cytiva, USA) was used for imaging analysis.
4
Western Blot Analysis of Striatal Protein Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the total protein lysates, the striatum tissues were homogenized using glass homogenizer with 10 volumes of RIPA buffer plus protease and phosphatase inhibitor cocktails and then the mixture was added with 4 × protein loading buffer (Invitrogen) with shaking and heating for 3 min. After that, the samples were centrifuged at 13, 000 rpm for 10 min at 4 °C and the supernatant was preserved.
The prepared protein extracts were size fractioned by 4 to 12% NuPAGE Bis-Tris gel electrophoresis (Invitrogen) using MES running buffer (Invitrogen). After transfer to the nitrocellulose membranes using Transfer Cell (Bio-Rad), the membranes were blocked with Odyssey Blocking Buffer (LI-COR) and probed overnight with the appropriate dilutions of the primary antibodies. Incubation with the IRDye-labeled secondary antibodies (LI-COR, 1:10000) was performed for 1 h at room temperature. The protein bands of interest were visualized with Odyssey CLx Infrared Imaging Studio. The band intensity was quantified using ImageJ.
The prepared protein extracts were size fractioned by 4 to 12% NuPAGE Bis-Tris gel electrophoresis (Invitrogen) using MES running buffer (Invitrogen). After transfer to the nitrocellulose membranes using Transfer Cell (Bio-Rad), the membranes were blocked with Odyssey Blocking Buffer (LI-COR) and probed overnight with the appropriate dilutions of the primary antibodies. Incubation with the IRDye-labeled secondary antibodies (LI-COR, 1:10000) was performed for 1 h at room temperature. The protein bands of interest were visualized with Odyssey CLx Infrared Imaging Studio. The band intensity was quantified using ImageJ.
5
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Using BCA protein assay (Sigma Aldrich), equal amounts of proteins were denatured with 1X protein loading buffer (ThermoFisher Scientific) at 100°C for 10 minutes. Samples were electrophoresed in an SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore) by semidry electroblotting (Bio-Rad) as described.22 (link) Membranes were probed with primary antibodies for HRas (1:200; sc-520, Santa Cruz Biotechnology), c-Myc (1:200; sc-789, Santa Cruz Biotechnology), p53 (1:500; sc-126, Santa Cruz Biotechnology), COX2 (1:1000; clone CX229, Caymann Chemicals), E6 (sc-460, Santa Cruz Biotechnology), E1A (1:1000, clone M73, Millipore), ATF4 (1:250, sc-200, Santa Cruz Biotechnology), CHOP (1:1000, sc-7351, Santa Cruz Biotechnology), SCD1 (1:300, sc30435, goat polyclonal, Santa Cruz), β-actin (1:1000, A1978, Sigma-Aldrich), BiP (1:250, sc-1050, Santa Cruz Biotechnology), HA (1:1000, H6908 Sigma), SCD-5 (AP53809PU-N, 2BScientific Ltd), ATF-3 (sc-188(C-19), Santa Cruz), and against GAPDH (1:1500, sc-32233, Santa Cruz). The antibody against Bap31 was kindly provided by Dr Gordon Shore. Antibody binding was detected by enhanced chemiluminescence (Thermo Sientific) and visualised on film.
6
Western Blot Protein Analysis Protocol
7
Chondrocyte Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The reagent RIPA Lysis Buffer (Thermo Fisher Scientific) was used to extract the total protein of the cultivated chondrocyte or isolated cartilage tissue. RIPA was supplemented with protease inhibitor PMSF. Protein concentration was determined by a BCA kit (Thermo Fisher Scientific). Protein was mixed with protein loading buffer (Thermo Fisher Scientific) and loaded to a 12% SDS-PAGE gel for electrophoresis. Afterward, the gel was transferred to PVDF membrane (Thermo Fisher Scientific). After a blocking step with a blocking buffer, the membrane was incubated with primary monoclonal antibodies at 4 °C overnight. After washing, the membrane was incubated with secondary antibody. Finally, signal was detected by an ECL method. The primary antibodies used in this study includes anti-Aggrecan (1:1000, Abcam), anti-Collagen II (1:1000, Abcam), anti-β-actin (1:3000, Abcam), anti-ADAM8 (1:1000, Abcam), anti-p-ERK (1:1000, Abcam), anti-ERK (1:2000, Abcam), anti-p- NF-κB p65 (1:1000, Abcam), anti-NF-κB p65 (1:2000, Abcam), anti-MMP9 (1:1000, Abcam), anti-Notch1 (1:1000, Abcam), anti-Hes1 (1:2000, Abcam). All the primary antibodies were incubated for 4 h at 37 °C. The HRP-conjugated secondary antibody (1:5000, Sigma-Aldrich) was incubated for 2 h at RT.
8
Immunoblotting Analysis of αSMA and HSP90
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed twice with PBS, collected with RIPA Buffer (Sigma-Aldrich) and sonicated (Sonifier 250, Branson Ultrasonics Corporation, Danbury, CT, United States) prior loading on gels. The protein concentration was determined by Pierce BCA Protein Assay (Thermo Fisher Scientific). Protein Loading Buffer and NuPAGE reducing agent (Thermo Fisher Scientific) were added in a 1:5 and 1:10 ratio, respectively. Samples were incubated at 70°C for 10 min, separated by SDS-PAGE and blotted on a nitrocellulose membrane. Immunodetection of αSMA and HSP90 was performed using Anti-alpha smooth muscle actin (1:200, cat # ab5694; Abcam, RRID:AB_2223021 ) and HSP90 α/β antibodies (F-8) (1:500, cat. # sc-13119; Santa Cruz Biotechnology, Dallas, TX, United States, RRID:AB_675659 ) in combination with fluorescent labeled secondary antibodies [1:20,000; IRDye® 800CW Donkey anti-Rabbit (cat. # 926-32213, RRID:AB_621848 ), IRDye® 680RD Donkey anti-Mouse (cat. # 926-68072, RRID:AB_10953628 )] diluted in Intercept® Blocking Buffer (cat. # 927-60001) (all from LI-COR Biosciences, Lincoln, NE, United States). Primary antibodies were incubated over night at 4°C, secondary antibodies for 1 h at RT, respectively. Membranes were analyzed with the Odyssey Fc Imaging System (LI-COR Biosciences).
9
Western Blot Analysis of PDCoV Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The purified PDCoV particles were resuspended in 1 × PBS and 5 × Protein Loading Buffer (Thermo Fisher Scientific, USA). Samples were separated by 12% SDS-PAGE. The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane that was subsequently blocked with Tris-buffered saline (TBS) containing 3% bovine serum albumin (BSA) at room temperature for 2 h and then incubated overnight at 4 °C with a hyperimmune rabbit anti-PDCoV serum or an anti-NS6-pt antibody. The blots were then incubated with Goat horseradish peroxidase (HRP) conjugated anti-rabbit IgG (Thermo Fisher Scientific, USA).
For Western blot analysis, PDCoV-infected cells were lysed in lysis buffer (25 mM Tris-HCl, 200 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 25 mM β-glycerophosphate, 1% NP40, and protease cocktail (Biotool, Houston, TX). Samples were resolved on SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane that was subsequently blocked with Tris-buffered saline (TBS) containing 3% bovine serum albumin (BSA) overnight at 4 °C. Proteins were detected using the anti-N mAb or anti-M pAb at 1:1000 dilution followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5000 dilution; Thermo Fisher Scientific).
For Western blot analysis, PDCoV-infected cells were lysed in lysis buffer (25 mM Tris-HCl, 200 mM NaCl, 10 mM NaF, 1 mM Na3VO4, 25 mM β-glycerophosphate, 1% NP40, and protease cocktail (Biotool, Houston, TX). Samples were resolved on SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane that was subsequently blocked with Tris-buffered saline (TBS) containing 3% bovine serum albumin (BSA) overnight at 4 °C. Proteins were detected using the anti-N mAb or anti-M pAb at 1:1000 dilution followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:5000 dilution; Thermo Fisher Scientific).
10
Detailed CCL4-induced Liver Fibrosis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CCL4 was purchased from Sigma (#488488). Olive oil (#PHR2902) from Merck. BCA protein quantitative kit Sirius Red reagent (#365548) and Masson (#1.00485) were purchased from Merck Biotechnology Co., Ltd. Xylene and alcohol were purchased from Huacheng Biology Co., Ltd. BSA powder was purchased from Solarbio Co., Ltd. Dulbecco's modified basic medium (DMEM, #11965118) was from Gibco (USA). α-SMA (Cat No. 14395-1-AP, 1 : 1000 dilution), TNF α (Cat No. 60291, 1 : 1000 dilution), TGF-β1 (Cat No: 21898-1-AP, 1 : 1000 dilution), and anti-fibronectin (Cat No. 66042-1-Ig, 1 : 500 dilution) were purchased from Proteintech Company (Wuhan, China). Anticollagen (ab270993, 1 : 800 dilution) was from Abcam (Cambridge, UK).CoraLite488-conjugated goat anti-rabbit IgG (H+L) was from Proteintech Company (Wuhan, China). Mouse IL-1β ELISA kit (#PI301), mouse TNF-α ELISA kit (#PT512), and mouse IL-6 ELISA kit (#PI326) were purchased from Beyotime Co., Ltd. Western blot and IP cell lysate were purchased from Huacheng Co., Ltd. Protein-loading buffer and protein prestaining marker were purchased from Thermo Fisher Technology (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!