After 48 h of cell transfection, the total protein was extracted from protein lysate. The sample and loading buffer were mixed according to the corresponding ratio, and then the sample was denatured in a boiling water bath. Each lane was loaded with an equal amount of protein (30 μg). After electrophoresis, the proteins were transferred to PVDF membranes. After blocking with 5% skimmed milk powder, blots were incubated in the corresponding primary antibody (1:1000, ab112543, Abcam) followed by the secondary antibody the next day. Then, ECL developer solution was added in a dark environment, and the blot was exposed using a gel imager. The final result is expressed as a target strip. The ratio of the optical density of the target band to that of the internal control GAPDH (1:2000, AF1186, Biyuntian) was reported as the protein expression level.
Af1186
Manufactured by Beyotime
Sourced in China, United States, United Kingdom
The AF1186 is a laboratory instrument designed for precise temperature control and monitoring. It is capable of maintaining a stable temperature environment for various applications within a research or testing setting.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
A0208, by Beyotime (3 mentions)
Ab112543, by Abcam (2 mentions)
Ab125066, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ab205921, by Abcam (1 mentions)
Sc 2357, by Santa Cruz Biotechnology (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Enhanced chemiluminescence detection kit, by Beyotime (1 mentions)
Af0111, by Beyotime (1 mentions)
Bca method, by Thermo Fisher Scientific (1 mentions)
Am071, by Beyotime (1 mentions)
Af1051, by Beyotime (1 mentions)
Af5860, by Beyotime (1 mentions)
Aj518, by Beyotime (1 mentions)
24 protocols using af1186
1
Western Blot Quantification Protocol
2
Western Blot Protein Analysis
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After 48 hours of cell transfection, the total protein was extracted with protein lysate, the sample and loading buffer were mixed according to the corresponding ratio, and then the sample was denatured in a boiling water bath. Each lane was loaded with an equal amount of 30µg. After the cell electrophoresis, the protein was transferred to the PVDF membrane. After blocking with 5% skimmed milk powder, incubate the corresponding primary antibody (1:1000, ab112543, Abcam), incubate the secondary antibody the next day, and add ECL developer solution in a dark environment and expose in a gel imager. The nal result is expressed as a target strip. The ratio of the belt's optical density to the internal control GAPDH (1:2000, AF1186, Biyuntian) was used as the protein expression level.
3
Protein Expression Profiling of Colorectal Cancer Cells
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Colorectal cancer cells of RKO, DLD-1, Caco-2, HCT8 at the logarithmic growth phase were lysed on ice for 30 min with RIPA (Beyotime Biotechnology, China). The cell lysates were centrifuged, and the protein concentration was detected by Coomassie Blue staining. Approximately 50 μg of cell lysates were electrophoresed on 8% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with antibodies of Rabbit anti-human PD-L1 (1:1000, ab205921, Abcam, UK) and GAPDH (1:1000, AF1186, Beyotime Biotechnology, China) at 4 °C overnight. After incubation with mouse anti-rabbit IgG-HRP antibody (1:1000, sc-2357, Santa Cruz Biotechnology, USA), the membranes were visualized using western blotting ECL reagent solution (Shanghai Share Biotechnology Co., Ltd., China).
4
Western Blot Analysis of Cell Cycle Regulators
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RIPA lysis buffer was used to prepare total lysate. A bicinchoninic acid (BCA)
method (Thermo Fisher Scientific) was used to measure protein concentration.
Equal amount of protein samples (20 μg) were added to electrophoresis chambers
and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
and then transferred onto polyvinylidene fluoride membranes. Subsequently, these
membranes were blocked with 5% skimmed milk for an hour at room temperature, and
incubated with antibodies against PIMREG (1:1000, ab118102, Abcam, Cambridge,
UK), CDK1 (1:1000, AF0111, Beyotime, Nantong, Jiangsu, China), phosphor (p)-CDK1
(Thr14, 1:1000, AF5758, Beyotime), CCNE1 (1:1000, AF6384, Beyotime), CCNB1
(1:1000, AF6627, Beyotime) and GAPDH (1:1000, AF1186, Beyotime) at 4°C for 24
hours. Followed by washing in Tris-buffered saline with 0.1% Tween-20 thrice,
the membranes were subjected to the corresponding horseradish
peroxidase-conjugated secondary antibodies and incubated for another 1 hour.
Signals were visualized by the enhanced chemiluminescence detection kit
(Beyotime), and images of the blots were analyzed using the ImageJ software
(NIH, Bethesda, MD, USA).
method (Thermo Fisher Scientific) was used to measure protein concentration.
Equal amount of protein samples (20 μg) were added to electrophoresis chambers
and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
and then transferred onto polyvinylidene fluoride membranes. Subsequently, these
membranes were blocked with 5% skimmed milk for an hour at room temperature, and
incubated with antibodies against PIMREG (1:1000, ab118102, Abcam, Cambridge,
UK), CDK1 (1:1000, AF0111, Beyotime, Nantong, Jiangsu, China), phosphor (p)-CDK1
(Thr14, 1:1000, AF5758, Beyotime), CCNE1 (1:1000, AF6384, Beyotime), CCNB1
(1:1000, AF6627, Beyotime) and GAPDH (1:1000, AF1186, Beyotime) at 4°C for 24
hours. Followed by washing in Tris-buffered saline with 0.1% Tween-20 thrice,
the membranes were subjected to the corresponding horseradish
peroxidase-conjugated secondary antibodies and incubated for another 1 hour.
Signals were visualized by the enhanced chemiluminescence detection kit
(Beyotime), and images of the blots were analyzed using the ImageJ software
(NIH, Bethesda, MD, USA).
5
Quantifying GPX4 in Septic Mouse Liver
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Liver tissue samples from septic mice were obtained and ground, and protein extraction and western blot analysis were performed as described previously21 (link). The primary antibodies included anti-GPX4 antibody (ab125066, 1:3000) from Abcam (Cambridge, UK) and anti-GAPDH (AF1186, 1:3000) from Beyotime Biotechnology (Shanghai, China). ImageJ software was applied to quantify the band intensity, and the ratio of the target protein to GAPDH was used to display the results.
6
Protein Expression Analysis in Cell and Tissue
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The L02 cells and liver tissue samples were prepared as described above, and total protein was extracted. Western blotting was used to detect the target proteins of each sample, such as phospho-ERK1/2 (p-ERK1/2) (AM071, Beyotime), ERK1/2 (AF1051, Beyotime), phospho-p38 MAPK (Thr180) (p-p38) (AF5884, Beyotime), p38 MAPK (p38) (AF7668, Beyotime), phospho-JNK1/2 (Thr183/Tyr185) (p-JNK) (AF5860, Beyotime), JNK (AJ518, Beyotime), GAPDH (AF1186, Beyotime), phospho-AMPKα (Thr172) (p-AMPKα) (2535T, CST), AMPKα (t-AMPK) (2532S, CST), phospho-ACC (Ser79) (p-ACC) (3661S, CST), and ACC (t-ACC) (3676T, CST). The target protein bands of western blots were analyzed and quantified. The levels of p-AMPKα, p-ACC p-ERK1/2, p-p38 and p-JNK were normalized to those of AMPKα, ACC, ERK1/2, p38 and JNK, respectively, and are presented as the fold change compared to the control treatment.
7
Western Blot Quantification of GAPDH and PARP
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Cells were plated in 6-well plates, cultured for 24 h with various concentrations of NC and BF, centrifuged, and then washed three times with 1× PBS. One hundred microliters of cell lysate was then added to each well, and lysed on ice for 10 min. The supernatant was taken and used to quantify the expression levels of GAPDH (Beyotime, AF1186, 1:2000) and PARP (Beyotime, AF1657, 1:5000). Western blot analysis typically includes several steps, including protein separation based on molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretic transfer (Bole, PowerPac Basic) of proteins from the SDS-PAGE gels to polyvinylidene difluoride (PVDF) membranes (Millipore, ISEQ00010), and blocking the membrane with 5% skim milk. Transfer the membrane to the primary antibody solution, shake at 4 °C overnight, and then wash 3 times with PBST for 3–5 min. The membrane was then transferred to the secondary antibody (Abcom, ab6721, 1:10000) solution and shaked at room temperature for 1 h. Wash the membrane 3 times with PBST for 3–5 min.
8
Protein Extraction and Western Blot Analysis
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Total protein was extracted using protein lysis solution (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Then Bicinchoninic Acid (BCA) protein concentration determination kit (Beyotime, Shanghai, China) was used to quantify the extracted protein. After adding sodium dodecyl sulfate (SDS)-loading buffer, it was placed in boiling water at 98°C for 5 min to denature. The sample was separated by 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Then the protein was transferred to polyvinylidene fluoride (PVDF, Millipore, Bedford, USA) membrane by Western electrophoresis. The membrane was incubated in 5% skimmed dry milk for 2 h. After incubation, they were incubated overnight with primary antibody: Bcl-2 (1RV 1000, AF0060, Beyotime, Shanghai, China), Bax (1RV 1000, AF1270, Beyotime, Shanghai, China), caspase1 (1RV 1000, AF1681, Beyotime, Shanghai, China), IL-1 β (1RV 1000, AF7209, Beyotime, Shanghai, China), NLRP3 (1RV 1000, AF2155, Beyotime, Shanghai, China) and GAPDH (1RV 1000, AF1186, Beyotime, Shanghai, China). The corresponding second antibody was horseradish peroxidase labeled goat anti-rabbit IgG (H+L) (A0208,1VO1000, Beyotime, Shanghai, China). Finally, the PVDF films were treated with Clarity Western ECL substrate (1705061 USA) and observed by chemiluminescence imaging system (Bio-Rad, USA).
9
Protein Quantification and Western Blot Analysis
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Protein was extracted from whole cell lysates and quantified using a BCA Protein Quantification kit (P0012S; Beyotime) as described previously 22 (link). Equal amounts of protein (10 µg) were separated using 10% SDS-PAGE. The blotted polyvinylidene (PVDF) membranes were incubated with antibodies against C1GalT1 (ab57492; Abcam, 1:1000), GAPDH (AF1186;Beyotime,1:2000), β1-integrin(ab24693; Abcam, 1:1000), Try397 pFAK (sc81493; Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:800), Ser 473 pAkt (sc293125; Santa Cruz Biotechnology, 1:800) and biotinylated Jacalin (B1115; Vector Laboratories, Burlingame, CA, 1:1000). The membranes were probed with HRP-conjugated anti-mouse (A0126; Beyotime, 1:2000) or anti-rabbit (A0108; Beyotime, 1:2000) secondary antibodies or Streptavidin (A0303; Beyotime, 1:2000). Protein bands were further visualized using ECL reagents of Pierce (Thermo Fisher Scientific, Rockford, IL, USA).
10
Western Blot Analysis of NF-κB Signaling
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The DCs were lysed by RIPA buffer mixed with protease inhibitors. Western blotting was performed according to the method described previously (Zhang et al., 2017 (link)). Antibodies of GAPDH (AF1186; Beyotime), p65 (AF1234; Beyotime), p-p65 (AF5875; Beyotime), and IκBα (AF5204; Beyotime) were used as the primary antibodies. The horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody was used as secondary antibody (A0208; Beyotime). Protein expression levels were normalized to GAPDH.
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