Active caspase-3 and MMP-3 was detected by Western blot with β-actin as an internal control. Cells were treated with various concentrations (30, 60, and 90 μg/ml) for 24 h, and then we collected the cells. This was accomplished by performing the following steps: (1) Completely lyse each sample by cell lysis buffer, then centrifuge them at 2000 r/min for 10 min. (2) Gather 100 μl of supernatant and add 20 μl lauryl sodium sulfate buffer (6×), then degeneration by boiling water bath for 10 min. (3) Separate protein by electrophoretic with 10% SDS-polyacrylamide gelatin, and then transfer samples to nitrocellulose membrane and confine for 1 h. (4) Add primary antibody of caspase-3 (Bioworld, diluted to 1: 500) and β-actin (Proteintech, diluted to 1:2000) at 37°C for an hour. (5) Second antibody (anti-rabbit IgG) marked with horse radish peroxidase (HRP) was added, at 37°C for 1 h. (6) Scan and analyze the result with digital gel imaging (Chemi Imager 4000, Alpha Innotech US).
Chemiimager 4000
Manufactured by Bio-Techne
Sourced in United States
The ChemiImager 4000 is a lab equipment product from Bio-Techne. It is a multi-function imaging system designed for a range of applications, including western blotting, fluorescence, and chemiluminescence imaging.
Automatically generated - may contain errors
Lab products found in correlation
Ab8227, by Abcam (2 mentions)
Western blue stabilized substrate for alkaline phosphatase, by Promega (2 mentions)
β actin, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ab6671, by Abcam (1 mentions)
Ab6199, by Abcam (1 mentions)
Ab16502, by Abcam (1 mentions)
Ab152, by Merck Group (1 mentions)
Abn100, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Ecl kit, by Santa Cruz Biotechnology (1 mentions)
Sc 81178, by Santa Cruz Biotechnology (1 mentions)
Anti lc3, by Santa Cruz Biotechnology (1 mentions)
Anti mapk1, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Nitrocellulose membrane, by Abcam (1 mentions)
Bca method, by Biocolor (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
11 protocols using chemiimager 4000
1
Caspase-3 and MMP-3 Protein Detection
2
Evaluating Neuroprotective Mechanisms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Active caspase-3, PARP, Akt, and p-Akt were detected by Western blot with GAPDH as a control. Human NPCs were divided into four groups: control, TNF-α (100 ng/mL), TNF-α (100 ng/mL) with pretreated 17β-estradiol (10 um/L), TNF-α (100 ng/mL) with pretreated 17β-estradiol (10 um/L) and MK2206 (10 um/L, inhibitor of PI3K/AKT pathway) for 24 hours, and then the cells were collected. This was accomplished by performing the following steps: (1) Complete lyses of each sample by cell lysis buffer, then centrifugation at 11,000 rpm for 15 minutes. (2) Collected 100 μL of supernatant and add 20 μL lauryl sodium sulfate buffer (6×), then degenerated by boiling water bath for 5 minutes. (3) Separated protein by electrophoretic with 12% SDS-polyacrylamide gelatin under 110 mV, and then transfer samples to nitrocellulose membrane and confined for 2 hour with 200 mA. (4) Added primary antibody of Akt, p-Akt, PARP, active caspase-3 (Bioworld, diluted to 1: 1,000), and GAPDH (Proteintech, diluted to 1: 2,000) at 37°C for one hour. (5) Antibody (anti-rabbit IgG) marked with horse radish peroxidase (HRP) was added at 37°C for one hour. (6) Scan and analyze the result with digital gel imaging (Chemi Imager 4000, Alpha Innotech USA).
3
Western Blot Analysis of NF-κB, NGF, and TNF-α
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein isolation and Western blot were conducted as previously reported [9 (link)]; a total of 20 mg protein was loaded for each sample. The primary antibodies used in Western blot were nuclear factor-κB (NF-κB, 1:500, ab16502; Abcam, Cambridge, MA, USA), NGF (1:300, ab6199; Abcam), TNF-α (1:500, ab6671; Abcam), and β-actin (1:1,000, ab8227; Abcam). After incubation with the secondary antibody, the resulting images were analyzed with ChemiImager 4000 (Alpha Innotech Corp., San Leandro, CA, USA) to determine the integrated density value of each protein band.
4
Cellular Lysis and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were lysed in the lysis buffer (150 mM NaCl, 0.1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM-Tris, 1mM-dithiothreitol, 5 mM-Na3VO4, 1mM-phenylmethylsulfonyl fluoride, 10 μg/ml trypsin, 10 μg/ml aprotinin, 5μg/ml leupeptin; pH 7.4). After incubation for 30 min at 4°C, the sample was centrifuged at 15,000 g for 8 min at 4°C, and the supernatant was collected as whole cell lysate and stored at -80°C until use. Equivalent amounts of proteins were separated on 10% SDS–PAGE gels and transferred to a nitrocellulose membrane. Western blot analyses were performed using LC3-II, AMPK, p-AMPK, p38 MAPK, p-p38 MAPK, Akt, p-Akt, mTOR, p-mTOR, p70S6K, p-p70S6K, 4E-BP-1, p-4E-BP-1, and β-actin antibodies. The membrane was incubated with the secondary alkaline phosphatase-conjugated IgG and detected using the Western Blue Stabilized Substrate for alkaline phosphatase (Promega, USA). The bands were analyzed using ChemiImager 4000 (Alpha Innotech, USA).
5
Protein Quantification and Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein isolation and Western blot were conducted as previously reported (11 (link)). A total of 20 mg protein was loaded for each sample. The primary antibodies used in Western blot were anti-tyrosine hydroxylase (TH, 1:500, AB152, Millipore, Bedford, MA, USA), anti-vesicular acetylcholine transporter (VAChT, 1:500, ABN100, Millipore, Bedford, MA, USA) and β-actin (1:1,000, ab8227; Abcam). After incubation with the secondary antibody, the resulting images were analyzed with ChemiImager 4000 (Alpha Innotech Corp., San Leandro, CA, USA) to determine the integrated density value of each protein band.
6
Protein Expression Analysis by Western Blot
7
Quantifying MAPK1 and LC3 Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies used in these experiments were anti-MAPK1 (1:1000, Santa Cruz
Biotechnology), anti-LC3 (1: 1000, Santa Cruz Biotechnology), and β-actin protein (1:
1000; sc-81178; Santa Cruz Biotechnology). Cells from each group were digested (50 mM
Tris–HCl (pH 6.8), 10 mM ethylenediaminetetraacetic acid (EDTA), 2% sodium dodecyl sulfate
(SDS), 5 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride) and then the
supernatants were collected after centrifugation of 15,000 × g for 1 h.
Cell lysate (100 ml) was collected to perform protein quantification using the Bradford
method. Equal amounts of protein (20 mg) from each cell sample were separated by SDS-
polyacrylamide gel electrophoresis (PAGE) on an 8% polyacrylamide gel and transferred to
polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA, USA). This membrane was
incubated overnight with a primary antibody (dilution 1:1000) at 41°C, then incubated with
a horse radish peroxidase-conjugated secondary antibody (Zymed Laboratory, San Francisco,
CA, USA) for 1 h at room temperature. Detection of reactive antigens was performed using
an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology). The resulting image
was analyzed with ChemiImager 4000 (Alpha Innotech, San Leandro, CA, USA) for protein band
densitometry.
Biotechnology), anti-LC3 (1: 1000, Santa Cruz Biotechnology), and β-actin protein (1:
1000; sc-81178; Santa Cruz Biotechnology). Cells from each group were digested (50 mM
Tris–HCl (pH 6.8), 10 mM ethylenediaminetetraacetic acid (EDTA), 2% sodium dodecyl sulfate
(SDS), 5 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride) and then the
supernatants were collected after centrifugation of 15,000 × g for 1 h.
Cell lysate (100 ml) was collected to perform protein quantification using the Bradford
method. Equal amounts of protein (20 mg) from each cell sample were separated by SDS-
polyacrylamide gel electrophoresis (PAGE) on an 8% polyacrylamide gel and transferred to
polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA, USA). This membrane was
incubated overnight with a primary antibody (dilution 1:1000) at 41°C, then incubated with
a horse radish peroxidase-conjugated secondary antibody (Zymed Laboratory, San Francisco,
CA, USA) for 1 h at room temperature. Detection of reactive antigens was performed using
an enhanced chemiluminescence (ECL) kit (Santa Cruz Biotechnology). The resulting image
was analyzed with ChemiImager 4000 (Alpha Innotech, San Leandro, CA, USA) for protein band
densitometry.
8
Exosome Protein Profiling via Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NPCs and exosomes were lysed, and the protein concentration was measured by BCA. The lysates were centrifuged, then subjected to SDS-PAGE and transferred to PVDF membranes, and the membranes were blocked with 5% skim milk for 1 h at room temperature. After washing with 1xTBST buffer, the primary antibody was added and incubated at 4°C overnight. Then, the membrane was washed 3 times in 1×TBST buffer and the second antibody was added and incubated at room temperature for 2 h. The protein expression level was analyzed by digital gel imaging (Chemi Imager 4000, Alpha Innotech USA). Human serum exosomes (Umibio Co., Shanghai, China) served as the positive control protein (PC).
9
Western Blot Analysis of Colon Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen colon samples were ground into powder in liquid nitrogen and lysed in ice-cold NP-40 lysis buffer containing 1 mM of protease inhibitor PMSF for 30 min. Colon tissue homogenate was centrifuged at 16,000×g for 20 min at 4°C, the supernatants were collected for Western blot analysis. Equal amounts of proteins (60 μg) were subsequently separated with SDS–polyacrylamide gel electrophoresis and transferred to PVDF membranes. After blocking with 5% skimmed milk at room temperature for 1 h, the membranes were incubated with primary antibodies at 4°C overnight, washed with TBST, and incubated with HRP-conjugated secondary antibodies at room temperature for 1.5 h. The membranes were visualized with an enhanced chemiluminescence reagent. The relative densities of the individual bands were analyzed densitometrically using the ChemiImager 4,000 instrument (Alpha Innotech, USA).
10
Quantifying Protein Expression in Y79 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We grouped the Y79 cells as follows: ( The monolayer cultures were collected with cell scrapers and then lysed with 100 μL of cell lysis buffer on ice for 30 min. The cell lysates were centrifuged, and supernatants were collected. Total protein was prepared from each group. The protein concentrations in the supernatants were aliquoted and maintained using the BCA method (Biocolor, Shanghai, China) for further experiments. A total of 50 μg of protein per sample was electrophoresed with 10% polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Millipore, Billerica, MA). It was blocked with 5% skimmed milk for 1 h at room temperature, and the nitrocellulose membrane was treated with primary antibodies (Abcam, Cambridge) at 4°C. After washing, the membrane was incubated with secondary antibodies (PCNA). The membrane was immersed in enhanced chemiluminescence solution and then exposed to an X-ray film. After the hybridization of the secondary antibodies, the resulting images were analyzed using ChemiImager 4000 (Alpha Innotech Corporation, California, 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!