A total protein extraction kit (KeyGen Biotech, Nanjing, China) was used to extract total proteins. The procedures were followed according to the kit manual. Cell protein lysates were separated by 10% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany) using a standard wet transfer apparatus. Then, the membranes were blocked in 5% non-fat milk in Tris-buffered saline with 0.05% Tween 20 (TBST) at room temperature for 2 hr, incubated overnight with the primary anti-HSPA12B (1:1,000 dilution, ab116082; Abcam), anti-Bcl2 (1:1,000, ab32124; Abcam), anti-Bax (1:1,000, ab77566; Abcam), anti-cleaved Caspase-3 (1:1,000, ab2302; Abcam), or anti-GAPDH antibody (1:1,000 dilution, AF0006; Beyotime); anti-GAPDH was used as an internal control. The membrane was washed with TBST three times for 5 min per wash. Then, the PVDF membrane was incubated in blocking buffer containing the diluted secondary antibody at room temperature for 2 hr. Finally, protein bands were detected on FluorChem FC2 Imaging System (Alpha Innotech, San Leandro, CA, USA).
Ab77566
Manufactured by Abcam
Sourced in United States
Ab77566 is a laboratory equipment product. It is a 96-well microplate designed for use in various assays and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ab32124, by Abcam (4 mentions)
Ab2302, by Abcam (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab194583, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ab196495, by Abcam (2 mentions)
Ab116082, by Abcam (1 mentions)
Af0006, by Beyotime (1 mentions)
Fluorchem fc2 imaging system, by Bio-Techne (1 mentions)
Sds page, by Bio-Rad (1 mentions)
A total protein extraction kit, by Keygen Biotech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab97779, by Abcam (1 mentions)
Ab109199, by Abcam (1 mentions)
Ab85570, by Abcam (1 mentions)
Ab131442, by Abcam (1 mentions)
Image reader las 3000, by Fujifilm (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
10 protocols using ab77566
1
Western Blot Analysis of Protein Targets
2
Protein Quantification and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For protein preparation, cells were homogenized with cell lysis buffer (Cell Signaling, USA), and then the protein concentrations were measured by using the bicinchoninic acid assay (BCA) kit (Thermo, USA). Protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Next, the membranes were incubated in the primary antibodies (ADRA2A, 1:500, ab85570; Bax, 1:1000, ab77566; Bcl-2, 1:1000, ab32124; MMP-2, 1:1000, ab97779; MMP-9, 1:1000, ab38898; p16, 1:1000, ab220800; p21, 1:1000, ab109199; p53, 1:1000, ab131442, Abcam, UK; phospho-PI3K, 1:300, #9655; PI3K, 1:1000, #4257; phospho-AKT, 1:500, #13,038; AKT, 1:1000, #4685; phospho-mTOR, 1:500, #2971; mTOR, 1:1000, #2983, GAPDH, 1:1000, #2118, Cell signaling, USA) overnight at 4°C. Subsequently, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (Proteintech, USA) for 2 h at room temperature. The protein bands were visualized with ECL system (Thermo, USA) and then recorded by the LAS 3000 Image Reader (Fujifilm, Tokyo, Japan).
3
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted using the protein extraction kit (BestBio; BB-3101). The quantification of protein was analyzed using the Ultra Bradford Protein Assay Kit (Beyotime, P0006). Protein samples (30 µg) in each hole were electrophoresed with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Roche, 3010040001). The blots were sealed with 5% skim milk and incubated with primary antibodies at 4 ℃ for the night and secondary antibodies at room temperature for 1 h. Finally, the expression levels of proteins were detected by electrogenerated chemiluminescence (KALANG; KL-D3490). The primary antibodies included NF-κB/p65 (1:1,000, Abcam, ab16502), Bax (1:1,000, Abcam, ab77566), B-cell lymphoma protein 2 (Bcl-2; 1:1,000, Abcam, ab194583), Caspase-3 (1:1,000, Abcam, ab214430), and glyceraldehyde 3-phosphate dehydrogenase (1:2,000, Abcam, ab9482).
4
Immunofluorescence Analysis of Propofol-Induced Stress Response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ARPE-19 cells grown on glass coverslips were treated with propofol for different times and/or TG. After treatment, the cells were fixed with 4% paraformaldehyde for 15 min after washing in PBS for 3 times. Then, 1% BSA and 0.1% Triton X-100 in PBS were used to incubate the coverslips for 30 min. Then, the cells were treated with antibodies against p-eIF2α (1:400, 3597, Cell Signaling Technology), eIF2α (1:400, 5324, Cell Signaling Technology), ATF4 (1:400, 11815, Cell Signaling Technology), active caspase 12 (1:500, ab62484, Abcam), cleaved caspase 3 (1:400, 9964, Cell Signaling Technology), BiP (1:400, 3177, Cell Signaling Technology), CHOP (1:500, ab11419, Abcam), Bcl2 (1:400, 15071, Cell Signaling Technology), and Bax (1:500, ab77566, Abcam) for 2 h at room temperature. After the coverslips were washed in PBS, they were incubated with anti-rabbit or anti-mouse secondary antibody (1:400, 4412, Cell Signaling Technology). Cells nuclei were stained with DAPI (5 mg/ml) for 15 min. The coverslips were mounted with anti-fade mounting medium after washing in PBS. Images were obtained using a Zeiss Confocal Spectral Microscope (Carl Zeiss, Jena, Germany).
5
Protein Expression Analysis Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein content was extracted from the tissues and cells using Protein lysis buffer (C0481, Sigma, St Louis, MO). Following 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the proteins were transferred onto a nitrocellulose membrane. The membranes were subsequently incubated with the following antibodies to HK2 (dilution ratio of 1 : 1000, ab104836, Abcam), mTOR (dilution ratio of 1 : 2000, ab2732, Abcam), p-mTOR (dilution ratio of 1:1000, ab109268, Abcam), Beclin1 (dilution ratio of 1 : 500, ab114071, Abcam), Bax (dilution ratio of 1 : 1000, ab77566, Abcam), Bcl-2 (dilution ratio of 1 : 1000, ab692, Abcam), 4EBP1 (dilution ratio of 1 : 1000, ab32130, Abcam), p-4EBP1 (dilution ratio of 1:1000, ab47365, Abcam), LC3A/B (dilution ratio of 1 : 1000, ab128025, Abcam), MMP-9 (dilution ratio of 1 : 1000, ab73734, Abcam) and β-actin (dilution ratio of 1:500, Beijing Cwbiotech Co., Ltd., Beijing, China) overnight at 4°C. Afterwards, the horseradish peroxidase (HRP) -labeled goat anti-mouse or anti-rabbit IgG (SPA131 or SA27, dilution ratio of 1 : 500, Solarbio, Beijing, China) was incubated with the membrane at room temperature for 1.5 h. The relative expression of the proteins was measured as previously described 51 (link).
6
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed using RIPA lysis buffer (Beyotime) on ice and centrifuged (20 min, 14, 000g, 4°C) to extract total protein. Thereafter, the protein samples were denatured by heating at 100°C for 3–5 min. After measuring protein concentration with BCA protein assay kit (Abcam, Cambridge, UK), extracted protein samples (about 30 μg/lane) were separated using sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Sangon Biotech), and protein was then transferred to nitrocellulose membranes (Invitrogen). After blocking with 5% skim milk (Beyotime), the membranes were then incubated with the primary antibodies at 4°C for 12–16 h: B-cell lymphoma-2 (Bcl-2; ab194583, 1:500, Abcam), BCL2-associated X protein (Bax; ab77566, 1:1000, Abcam), SOX4 (ab70598, 1:500, Abcam), β-actin (ab227387, 1:5000, Abcam). Afterwards, these membranes were probed with corresponding secondary antibody (ab205718/ab205719, 1:4000, Abcam). Finally, the combined signals were detected by enhanced chemiluminescence reagent (Solarbio, Beijing, China). Quantification of protein levels was determined using ImageJ software. The protein abundance was normalized by β-actin. All experiments were repeated three times.
7
Western Blotting Procedure for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein used for Western blotting was extracted using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, P.R. China) supplemented with protease inhibitors (Roche, Guangzhou, P.R. China). The proteins were quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). The Western blot system was established using a Bio-Rad Bis-Tris Gel System according to the manufacturer’s instructions. Primary antibodies against YEATS4 (ab205018), GAPDH (ab128915), phosphorylated β-catenin (p-β-catenin, ab138378), β-catenin (ab6302), Bcl-2 (ab32124), Bax (ab77566), c-Myc (ab152146), CDK6 (ab151247), CDK4 (ab137818), and cyclin D1 (ab137875) (all from Abcam, Cambridge, MA, USA) were prepared in 5% blocking buffer. Primary antibodies were respectively incubated with the membrane at 4°C overnight, followed by wash and incubation with secondary antibodies marked by horseradish peroxidase for 1 h at room temperature. After rinsing, the polyvinylidene difluoride (PVDF) membrane carrying blots and antibodies were transferred into the Bio-Rad ChemiDoc™ XRS System, and then 200 μl of Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) was added to cover the membrane surface.
8
Investigating Cellular Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After treatment of each group of cells for 3 days, the collected cells were tested for expression of PI3K, AKT, mTOR, Bcl-2, Bax, etc., and were operated according to the previously described method. The antibodies used were as follows: anti-PTEN (ab32199, Abcam), anti-p53 (2542 CST), anti-PI3K (ab186612, Abcam), anti-p-PI3K (ab182651,Abcam), anti-AKT1/2/3 (ab179463, Abcam), anti-p-AKT(s473) (ab81283, Abcam), anti-mTOR (2983, CST), anti-p-mTOR(s2448) (5536,CST), anti-cyclin B1 (ab181593, Abcam), anti-Bcl-2 (ab32124, Abcam), anti-cyclin D1 (ab134175, Abcam), anti-Bax (ab77566, Abcam), anti-p21 (ab109520, Abcam), anti-Caspase-3 (ab32351, Abcam), anti- GSK-3β (ab75814, Abcam), anti-β-actin (ab8226, Abcam), and anti-p27 (ab32034, Abcam), anti-JNK1 (ab199380, Abcam), anti-ASK1 (ab45178, Abcam), anti-p38 (ab182453, Abcam), anti- phosphol-p38 (ab178867, Abcam). The β-actin was used as an internal reference for this study.
9
Apoptosis and Oxidative Stress Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA lysate (Beyotime, Shanghai, China) was used to obtain total proteins, 100 μg of which were segregated using SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. TBST containing 5% skim milk was used for membrane incubation for 1 h. Then, the membranes experienced incubation with primary antibodies including Anti-Bax antibody (ab77566, 1 μg/mL, Abcam, Cambridge, MA, USA), anti-Bcl-2 antibody (ab196495, 1 : 500, Abcam), anti-Caspase-3 antibody (ab13847, 1 : 500, Abcam), anti-Cleaved Caspase-3 antibody (ab2302, 1 μg/mL, Abcam), anti-Nrf2 antibody (ab137550, 1 : 500, Abcam), anti-Heme Oxygenase 1 antibody (ab13243, 1 : 2000, Abcam), anti-NQO1 antibody (ab2346, 0.3 μg/mL, Abcam), anti-ROCK1 antibody (EP786Y) (ab45171, 1 : 2000, Abcam), anti-ROCK2 antibody (ab71598, 1 μg/mL, Abcam), and anti-GAPDH (ab181603, 1 : 10000, Abcam) at 4°C overnight. The membranes were washed in TBST three times and incubated with anti-rabbit IgG H&L (HRP) secondary antibody (ab6721, 1 : 2000, Abcam) at room temperature for 1.5 h. After washing using TBST thrice, the membranes were subjected to color reaction by ECL Plus from Life Technology, and GAPDH was detected as control groups.
10
Western Blot Analysis of Extracellular Vesicle Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In Western blot, we used RIPA (Radio-Immunoprecipitation Assay) and PMSF (Phenylmethanesulfonyl fluoride, Sigma, USA) to get the protein of the cells and tissue from each group. After electrophoresis, the membranes were incubated with the primary antibodies, including anti-CD63 antibody (1:1000, abcam, USA, ab59479), anti-CD81 antibody (1:1000, abcam, USA, ab219209), anti-CD90 antibody (1:1000, abcam, USA, ab225), anti-TSG101 (1:500, abcam, USA, ab125011), anti-GRP94
(1:500, abcam, USA, ab2791), anti-Cleaved Caspase 3 (1:800, abcam, USA, ab2302), anti-BAX (1:800, abcam, USA, ab77566), anti-BCL-2 (1:800, abcam, USA, ab196495), anti-BAX (1:1000, abcam, USA, ab181602). Next, the membranes were washed by PBS, and incubated by the following secondary antibodies: Affinpure horseradish peroxidase (HRP) -conjugated goat anti-rabbit lgG (1:5000, Haoranbio, Shanghai, China, HSA0003) or anti-mouse lgG (HSA0001). Finally, the membranes were washed by PBS and the proteins were visualized by ECL solution. The data was quantified by ImageJ2X software.
(1:500, abcam, USA, ab2791), anti-Cleaved Caspase 3 (1:800, abcam, USA, ab2302), anti-BAX (1:800, abcam, USA, ab77566), anti-BCL-2 (1:800, abcam, USA, ab196495), anti-BAX (1:1000, abcam, USA, ab181602). Next, the membranes were washed by PBS, and incubated by the following secondary antibodies: Affinpure horseradish peroxidase (HRP) -conjugated goat anti-rabbit lgG (1:5000, Haoranbio, Shanghai, China, HSA0003) or anti-mouse lgG (HSA0001). Finally, the membranes were washed by PBS and the proteins were visualized by ECL solution. The data was quantified by ImageJ2X software.
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!