Proteins were extracted using radioimmunoprecipitation assay buffer (P0013B; Beyotime, Shanghai, China). The protein samples were first separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Immobilon, Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in TBST buffer (TBS containing 0.1% Tween-20) at room temperature for 1 h, and then incubated with one of the following primary antibodies at 4°C overnight: rabbit anti-BAX (1:5000; Proteintech), rabbit anti-Bak (1:1000; Proteintech), rabbit anti-Bcl-2 (1:2000; Proteintech), and rabbit anti-β-actin (1:1000; Proteintech). After washing, the membranes were incubated with the appropriate secondary antibody (anti-rabbit Ig-G, 1:2000; Proteintech) for 1 h. The immunoreactive proteins were quantified using the NIH ImageJ software. β-Actin was used as an internal control. The protein levels are expressed as protein/β-actin ratios.
Rabbit anti β actin
Manufactured by Proteintech
Sourced in United States, China
Rabbit anti-β-actin is a primary antibody that specifically recognizes the β-actin protein. It is commonly used in Western blotting and immunohistochemistry applications to detect the expression of β-actin, a widely expressed and highly conserved cytoskeletal protein.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Solarbio (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Mouse anti gfp, by Proteintech (2 mentions)
Mouse anti gapdh, by Proteintech (2 mentions)
Culture cell total protein extraction reagent, by Boster Bio (2 mentions)
Bs 0296g, by Bioss Antibodies (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Hrp conjugated goat anti rabbit igg, by Merck Group (2 mentions)
Hrp conjugated goat anti mouse igg, by Merck Group (2 mentions)
Mouse anti hpiv3, by Abcam (2 mentions)
Rabbit anti lc3b specific, by Proteintech (2 mentions)
Rabbit anti p50, by Proteintech (2 mentions)
Rabbit anti p62 sqstm1, by Proteintech (2 mentions)
Mouse anti dic, by Merck Group (2 mentions)
Rabbit anti lamp2, by Abcam (2 mentions)
Rabbit anti timp 1, by Proteintech (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Rabbit anti foxm1, by Proteintech (2 mentions)
Total protein extraction kit, by Solarbio (2 mentions)
35 protocols using rabbit anti β actin
1
Protein Extraction and Western Blot Analysis
2
Immunoblotting Quantification of JAK2/STAT3 Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins from samples (n = 4/group) were extracted in RIPA lysis buffer (Solarbio, China) with PMSF and a phosphatase inhibitor, and the protein concentration was assessed by BCA protein assay kit (Solarbio, China). Proteins were separated on a 7.5% sodium dodecyl sulfate-polyacrylamide electrophoresis gel (SDS-PAGE) and then transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in TBST for 1 h and incubated overnight at 4°C in primary antibodies: rabbit anti-JAK2 (1:2,000; Cell Signaling Technology, United States), rabbit anti-phospho-JAK2 (Y1007 + 1,008) (1:2,000; Abcam, United States), rabbit anti-STAT3 (1:2,000; Cell Signaling Technology, United States), rabbit anti-phospho-STAT3 (Tyr705) (1:2,000; Cell Signaling Technology, United States), rabbit anti-β-actin (1:4,000; Proteintech Group, China). Later, the membranes were washed in TBST and then cultured with anti-rabbit HRP-conjugated secondary antibody (1:4,000; Proteintech Group, China) for 1 h. Finally, the enhanced chemiluminescence assay kit (Proteintech Group, China) was used to visualize immunoblots, and the densities of the relative target proteins were measured using ImageJ. The β-actin was chosen as the internal reference control.
3
Immunoblotting Viral Particles and Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoblotting of viral particles, 2 × 1011 genome copies of virus were boiled for 10 min in 6X Laemmli buffer (50 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, 0.1% (w/v) bromophenol blue, 100 mM β-mercaptoethanol) before separating on 4–12% Bis-Tris polyacrylamide gradient gel (Invitrogen). For immunoblotting from HEK293T cells, whole cell lysates were prepared by lysing cells in RIPA buffer (50 mM Tris-HCl (pH 6.8), 150 mM NaCl, 0.5% (w/v) sodium deoxycholate, 1% (v/v) NP-40, 0.1% (w/v) SDS, supplemented with protease inhibitors (Roche)), supplemented with 6X Laemmli buffer and boiled for 5 min. HEK293T cell supernatants were filtered through a 0.45 µM filter, supplemented with 6X Laemmli buffer and boiled for 5 min. After PAGE, proteins were transferred to a Hybond ECL membrane (Amersham biosciences) using a semi-dry transfer system (Biorad). Mouse-anti-HIV-1capsid p24 was from AIDS reagents (183-H12-5 C), mouse-anti-firefly luciferase was from Santa Cruz Biotechnology (sc-57,604), mouse-anti-GFP was from Proteintech (66002-1-Ig), rabbit-anti-β-actin was from Proteintech (20536-1-AP) and were detected with goat-anti-mouse/rabbit IRdye 680/800CW infrared dye secondary antibody and membranes imaged using an Odyssey Infrared Imager (LI-COR Biosciences).
4
Western Blot Analysis of Mitochondrial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Specific proteins expressed in the mouse brain and neuron cell cultures were determined by western blotting using specific antibodies. Proteins (40 µg) were separated on 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and then transferred onto a nitrocellulose membrane. Nonspecific binding sites were blocked using 5% fat-free dried milk in 1 × TBST at 37 °C for 1 h. The membrane was incubated at 4 °C overnight with individual primary antibodies, including rabbit anti-OPA1 (Cell Signaling Technology, USA), rabbit anti-MFN1 (Proteintech, USA), mouse anti-MFN2 (Abcam, USA), rabbit anti-cytochrome c (Cell Signaling Technology, USA), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, USA), mouse anti-GAPDH (Proteintech, USA), rabbit anti-β-actin (Proteintech, USA), and rabbit anti-VDAC (Cell Signaling Technology, USA). After washing with TBST, the membrane was incubated with secondary antibodies, goat anti-mouse IgG or anti-rabbit IgG conjugated to horseradish peroxidase (1:10,000). The blots were visualized using a Chemiluminescent Imaging System (Tanon Science and Technology).
5
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein lysate was obtained from tissue by culture cell total protein extraction reagent (BOSTER, Wuhan, CHN) and mammalian tissue protein extraction reagent (BOSTER, Wuhan, CHN), with protease inhibitor and phosphatase inhibitor cocktail, respectively. Protein concentration was determined by a BCA protein analysis kit (BIOMED, Beijing, CHN). The primary antibodies included the following: rabbit anti-β-actin (#20536-1-AP; Proteintech, Wuhan, CHN), rabbit anti-ZO-1 (#21773-1-AP; Proteintech, Wuhan, CHN), rabbit anti-occludin (#27260-1-AP; Proteintech, Wuhan, CHN), rabbit anti-Claudin 1 (#bs-1428R; Bioss, Beijing, CHN), rabbit anti-NF-κB P65 (#bs-0465R; Bioss, Beijing, CHN), rabbit anti-phospho-NF-κB p65 (#3033T; CST, Danvers, MA USA), rabbit anti-p38 (#ab27986; Abcam, Cambridge, MA, USA), and rabbit anti-phospho-p38(#ab47363; Abcam, Cambridge, MA, USA). The secondary antibody used was goat anti-rabbit (#bs-0296G; Bioss, Beijing, CHN).
6
ORF142 Protein Detection in LSDV-infected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDBK cells infected or not infected with LSDV-WT were washed three times with cold PBS at specific time points and then harvested with lysis buffer containing 1% protease inhibitors (Beyotime). The protein concentration was determined with a BCA Protein Assay Kit (Beyotime). The cell extracts were resolved with sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Beyotime). Total ORF142 protein was detected with a rabbit anti-ORF142 antibody (stored in our laboratory), with rabbit anti-β-actin (Proteintech, Wuhan, China) as the loading control.
7
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A cell scraper was used to quickly collect cells from each group, and nuclear and cytoplasmic proteins were extracted according to the instructions of a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China, P0028). The protein concentrations were determined by the BCA method. Thirty micrograms of protein was added to a 10% SDS-PAGE gel and then transferred to polyvinylidene uoride membranes. The membranes were incubated with fast blocking solution and then incubated with the following primary antibodies overnight at 4°C: rabbit anti-NF-κB p65 (1:2000, Beyotime, Shanghai, China, AF0246), rabbit anti-ErbB4 (1:500, Abcam, United Kingdom, ab32375), rabbit anti-p-ErbB4 (1:500, Abcam, United Kingdom, ab109273), mouse anti-Bax (1:1000, SANTA, Shanghai, China sc-7480), mouse anti-NRG1 (1:1000, SANTA, Shanghai, China, sc-393006), rabbit anti-histone H3
(1:1000, Beyotime, Shanghai, China, AF5614), rabbit anti-GAPDH (1:5000, Abcam, United Kingdom, AF1186), and rabbit anti-β-actin (1:5000, Proteintech, Chicago, USA, 66009-1-lg). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Pierce, Rockford, IL, USA) for 2 h at room temperature. The bands were detected by enhanced chemiluminescence (ECL). ImageJ software was used to assess the optical density values.
(1:1000, Beyotime, Shanghai, China, AF5614), rabbit anti-GAPDH (1:5000, Abcam, United Kingdom, AF1186), and rabbit anti-β-actin (1:5000, Proteintech, Chicago, USA, 66009-1-lg). The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Pierce, Rockford, IL, USA) for 2 h at room temperature. The bands were detected by enhanced chemiluminescence (ECL). ImageJ software was used to assess the optical density values.
8
Protein Expression Analysis in Renal Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein expression in renal tissue and cultured cells was analyzed by Western blotting. Cells were lysed in ice-cold lysis buffer, and protein concentration was determined using the BCA Protein Assay Kit (Beyotime). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and electrotransferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Germany). Subsequently, the membranes were blocked with 5% non-fat milk for 1 h at room temperature and probed with primary antibodies, such as rabbit-anti-BMP-7 (1:1000), rabbit-anti-Smad1 (1:1000; Proteintech), rabbit-anti-Smad5 (1:1000; Proteintech), rabbit-anti-Phospho- Smad1/5(Ser463/465) (1:1000, Cell Signaling Technology), rabbit-anti-E-cadherin (1:1000), rabbit-anti-Vimentin (1:1000; Santa Cruz), rabbit-anti-Collagen-III (1:1000), rabbit-anti-SnoN (1:1000), and rabbit-anti-β-actin (1:400; Proteintech), for 16 h at 4 °C. The signals were detected by chemiluminescence after incubation with appropriate secondary antibodies (). Images were acquired using the Bio-Rad gel imaging system (Bio-Rad, CA, USA), and the intensities of the immunoreactive bands were quantified using the Quantity One 4.6 software (Bio-Rad).
9
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Infected or transfected cells were harvested and lysed in cold TNE buffer (50 mM Tris-Cl [pH 7.4], 150 mM NaCl, 2 mM EDTA [pH 8.0], 0.1% 2-mercaptoethanol and protease inhibitor cocktail). After incubation on ice for 30 min, cell lysates were centrifuged at 13,000 rpm for 30 min at 4°C. The clarified supernatant was mixed with 5XSDS-PAGE loading buffer, boiled at 100°C for 10 min and then subjected to 12% sodium dodecyl sulfate-polyacrylamide gel. The primary antibodies used were as follows: mouse anti-HPIV3 (1:2500, Abcam), rabbit anti-β-actin (1:1000, Proteintech), mouse anti-Myc tag (1:2500, Santa Cruz), mouse anti-HA tag (1:10000, Sigma), mouse anti-Flag tag (1:2500, Sigma), mouse anti-cofilin (1:2500, Proteintech), and rabbit anti-p-cofilin (1:1000, Cell Signaling Technology). HRP-conjugated goat anti-mouse IgG (1:5000, Sigma) and HRP-conjugated goat anti-rabbit IgG (1:5000, Sigma) were used as secondary antibodies.
10
Immunoblotting Analysis of NLRC4 and Downstream Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were loaded (50 μg), separated by 6, 8, and 15% SDS–PAGE and then transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). The primary antibodies: rabbit anti-NLRC4 (1:800, Novus Biologicals, USA), mouse anti-pNLRC4 (1:400, ECM Biologicals, USA), mouse anti-RGS2 (1:500, Santa Cruz, USA), rabbit anti-Pro-caspase-1 (1:1000, Abconal, China), rabbit anti-Pro-IL-1β (1:1000, Abconal), rabbit anti-IL-18 (1:1000, Abconal, China), rabbit anti-cleaved caspase-1 (1:500, Cell Signaling, USA), rabbit anti-cleaved IL-1β (1:500, Affinity, USA), rabbit anti-cleaved IL-18 (1:200, R&D, USA), rabbit anti-TNF-α (1:500, Boster, China), rabbit anti-IL-6 (1:500, Boster, China), rabbit anti-LRRK2 (1:1000, Abcam, USA), rabbit anti-pLRRK2 s935 (1:500, Abcam, USA) and rabbit anti-β-actin (1:1000, Proteintech, USA). Secondary antibody (1:2000, Proteintech, USA)-linked horseradish peroxidase (HRP) was added to the bands for 2 h. ECL detection reagents (Thermo, USA) were used to visualize the bands. ImageJ software was used to determine the relative density of these proteins.
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!