Cells were lysed with RIPA buffer (Beyotime Institute of Biotechnology) containing protease inhibitors. Protein concentration was quantified using bicinchoninic acid protein assay (Pierce; Thermo Fisher Scientific, Inc.). Protein samples (10 µg) were separated on 12% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes (Roche, Basel, Switzerland). Membranes were incubated in 5% non-fat milk at 25°C for 1 h to block non-specific binding. Membranes were then membranes incubated overnight at 4°C with primary antibodies targeting phosphorylated (p-) Akt (cat. no. ab81283; 1:500; Abcam, Cambridge, MA, USA), total Akt (cat. no. ab18785; 1:500; Abcam), and β-actin (cat. no. ab8226; 1:500; Abcam). Membranes were washed three times with PBS, then samples were incubated with anti-rabbit or anti-mouse secondary antibodies purchased from Beyotime Institute of Biotechnology (cat. no. A0208/A0216; 1:1,000). Bands were visualized using enhanced chemiluminescence substrate (Thermo Fisher Scientific Inc.). Quantity One (version 4.0; Bio-Rad Laboratories, Inc.) was used for densitometric analysis.
Ab18785
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab18785 is a laboratory antibody product. It is designed for use in standard immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ab38449, by Abcam (14 mentions)
Ab182651, by Abcam (6 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Ab8245, by Abcam (4 mentions)
Ab32089, by Abcam (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Beyotime (3 mentions)
Ab81283, by Abcam (3 mentions)
Ab109268, by Abcam (3 mentions)
Ab9485, by Abcam (3 mentions)
Ab2732, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ab97051, by Abcam (2 mentions)
Ab38898, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Ecl kit, by Merck Group (2 mentions)
Ab134903, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
26 protocols using ab18785
1
Quantitative Western Blot Analysis
2
Cell Proliferation Assay and Protein Expression
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A total of 5 × 103 cells/well of each group were seeded into 96‐well plates (Corning), cultured at 37°C for 12 h with 5% CO2, and then 10 μl CCK‐8 solution (Beyotime) was added to each well. The cells were then cultured at 37°C with 5% CO2 for another hour. Then the value of optical density (OD) in each group was evaluated 450 nM using Microplate ReaderM200 PRO (Thermo Fisher Scientific). For western blot analysis, the following antibodies were used: Phospho‐pan‐AKT (Ser473; Affinity), Phospho‐PTEN (S380; Affinity), GAPDH (AF7021; Affinity), anti‐PI3K (ab191606; Abcam) and anti‐AKT (ab18785; Abcam).
3
Western Blot Analysis of Protein Expression
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After transfecting for 24 h, the cells were lysed with cell lysate, and the total protein was extracted and the protein concentration was determined with the BCA kit (Thermo, USA). The protein was then separated by 10% SDS-PAGE and transferred to the PVDF membrane. After being blocked by 5% skimmed milk at room temperature for 1 h, the primary antibody TCF-3 (ab229605, Abcam, UK), Akt (ab38449, Abcam), p-Akt (ab18785, Abcam), mTOR (ab2732, Abcam), p-mTOR (ab109268, Abcam), and β-actin (No.66009–1, Proteintech, USA) were added and incubated overnight at 4°C. After washing the membrane 3 times, the secondary antibody was added and incubated for 1 h at room temperature. After washing the membrane another 3 times, a chemiluminescence reagent (Gibco, USA) was added to develop the protein and placed in the gel imaging system to collect the image. We employed Image J software to analyze the gray level of the protein band and selected β-actin as the internal reference to calculate the protein relative expression.
4
Protein Expression Analysis of Apoptosis and UPR Pathways
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The total protein was extracted by the modified RIPA buffer (P0013B; Beyotime, Shanghai, China), and the concentration was quantitated by the BCA protein assay kit (P0010; Beyotime, Shanghai, China). Then, the total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Next, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, and then the membranes were blocked in 5% skim milk diluted with TBST for 1 h at room temperature and probed with primary antibodies, including MFG-E8 (R&D Systems, AF2805, 1 : 500), BCL2 (Abcam, ab117115, 1 : 500), BAX (Abcam, ab216494, 1 : 500), cleaved caspase-3 (Abcam, ab90437, 1 : 500), GRP-78 (Abcam, ab230508, 1 : 500), XBP-1 (Abcam, ab230508, 1 : 500), ATF-6 (Abcam, ab203119, 1 : 500), ATF-4 (Abcam, ab23760, 1 : 500), CHOP (Abcam, ab23760, 1 : 500), p-PI3K (Abcam, ab125633, 1 : 500), PI3K (Abcam, ab154583, 1 : 500), p-AKT (Abcam, ab18785, 1 : 1000), AKT (Abcam, ab28422, 1 : 500), and β-actin (Abcam, ab209857, 1 : 1000) at 4°C overnight. Then, the membranes were incubated with a secondary rabbit anti-rabbit antibody (1 : 1000) the next day at room temperature for 1 h. The ImageJ software was employed for quantitation of the immunoreactive bands.
5
Western Blot Analysis of EMT and Angiogenesis Markers
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Hep3b cells were washed in PBS and lysed using the protein extraction reagent RIPA (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration of proteins was measured by BCA kit (cat. no. ab102536; Abcam). Equivalent amounts of proteins (30 µg) from each sample were electrophoresed on SDS-polyacrylamide gel (SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane, blocked in 4% skim milk for 2 h at room temperature and incubated with the following specific primary antibodies: E-cadherin antibody (ab219332 1:1,000; Abcam), α-catenin antibody (ab51032 1:2,000; Abcam), N-cadherin (ab76011, 1:5000 dilution, Abcam), vimentin (ab92547 1:1,000; Abcam), p-PI3K (ab182651 1:1,000; Abcam), PI3K (ab227204 1:1,000; Abcam), p-AKT (ab38449, 1:500 dilution, Abcam), AKT (ab18785 1:1,000; Abcam), VEGF (ab214424 1:1,000; Abcam), VEGFR2 (ab221679 1:1,000; Abcam), Snail (ab53519 1:1,000; Abcam), Slug (ab27568 1:1,000; Abcam) and MPP9 (ab38898 1:1,000; Abcam) overnight at 4°C. β-actin (ab8277 1:1,000; Abcam) was used as internal reference. Then, the membranes were incubated in HRP-linked goat anti-rabbit IgG secondary antibody (ab97051; 1:10,000; Abcam) for 2 h at room temperature. Immunoreactivity was visualized by a colorimetric reaction using an ECL substrate buffer (EMD Millipore) and membranes were scanned with Gel Doz EZ imager (Bio-Rad Laboratories, Inc.).
6
Protein Expression Analysis of Pancreatic Cancer
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Protein samples isolated from indicated pancreatic cancer cells were loaded onto SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% skimmed milk, incubated with primary antibodies HK2 (Anti-HK2, 1:1000, ab209847, Abcam), PKM2 (Anti-PKM2, 1:1000, ab85555, Abcam), GUT1(Anti-GUT1, 1:1000, ab115730, Abcam), PTK2 (Anti-PTK2, 1:1000, ab40794, Abcam), AKT(Anti-AKT, 1:1000, ab18785, Abcam), p-AKT(Anti-AKT1-phospho-S473, ab81283, Abcam), mTOR(1:1000, 66888-1-Ig, Proteintech), p-mTOR(Anti-mTOR-phospho S2448, ab109268, Abcam), GAPDH (1:1000, 60004-1-Ig, Proteintech), followed by reaction with HRP-conjugated secondary antibodies (1:1000, Boster). ECL Western Blotting Substrate (Beyotime) was applied for Western blot band detection. Images were visualized under Biorad Imaging Systems.
7
Western Blot Analysis of Apoptosis Markers
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The protein samples were collected with the RIPA buffer (Beyotime, China). After that, BCA method was used for the detection of the concentration of these protein samples. Then, these protein samples were separated with 10% SDS-PAGE gel (Beyotime, China). Next, these proteins were transferred to the PVDF membranes (Millipore, USA). Then these membranes were blocked with the BSA (Beyotime, China) for 2 hours. Then, these membranes were incubated with the primary antibodies at 4°C overnight. The primary antibodies used in this research were Cleaved caspase-3 (Abcam, ab2302), Cleaved PARP (Abcam, ab32064), p-Akt (Abcam, ab18785), Akt (Abcam, ab64148) and GAPDH (Abcam, ab8245). All these primary antibodies were diluted with the BSA with the ratio (1:1000). On the second day, these cells were washed with the PBST and incubated with the second antibodies (Abcam, ab205718) for 2 hours in room temperature. The secondary antibodies were diluted with the BSA with the ratio (1:2000). Finally, the immunoreactive signals were measured with the Pierce Western Blotting Substrate (Thermo Fisher Scientific, USA). And the results were quantified and analyzed with Image J software (National Institutes of Health, USA) [21 (link)].
8
Western Blot Analysis of Oxidative Stress Markers
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Protein samples from RAW264.7 macrophages or lung tissues were washed with PBS, followed by the lysis on ice in lysis buffer containing phosphatase inhibitors and protease inhibitors. Next, the samples were centrifuged (12,000 × g) at 4°C for 15 min. Equal amounts of protein were separated on SDS-PAGE gel, which were then transferred onto polyvinylidene difluoride membranes. After that, 5% nonfat milk was used to block the membranes for 90 min at room temperature, followed by the incubation with primary antibodies at 4°C overnight. Then, the membranes were incubated with peroxidase-conjugated secondary antibodies at room temperature for 60 min. At last, the grey value of these protein bands was scanned and quantified using Image Lab software (BioRad, USA). The primary antibodies as well as their dilution ratio are displayed as follows: SOD1 (#ab183881, Abcam, 1 : 1000), SOD2 (#ab68155, Abcam, 1 : 1000), GPX4 (#ab252833, Abcam, 1 : 500), GAPDH (#ab8245, Abcam, 1 : 1000), phosphorylated-AKT (p-AKT) (#ab38449, Abcam, 1 : 500), AKT (#ab18785, Abcam, 1 : 500), p-Foxo1 (#ab259337, Abcam, 1 : 500), and Foxo1(#ab179450, Abcam, 1 : 500). The relative protein levels of genes were normalized with the level of GAPDH in the same group.
9
Protein Expression Analysis via Western Blot
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Total protein was extracted from whole-cell lysates using an RIPA lysis buffer kit (Thermo Scientific, Invitrogen). Equal amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, and the membranes were blocked with 5% nonfat milk for 1 h. The membranes were incubated with specific primary antibodies, including anti-IGF1R (ab182408, Abcam), anti-PI3K (ab32089, Abcam), anti-p-P13K (ab182651, Abcam), anti-AKT (ab18785, Abcam), and anti-p-AKT (ab38449, Abcam), at 4°C overnight. On the following day, the membranes were incubated with appropriate secondary antibodies at 37°C for 1 h. Protein bands were visualized using an enhanced chemiluminescence kit.
10
Protein Expression Analysis by Western Blot
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Cells were lysed in RIPA Lysis Buffer (Beyotin, China) with PMSF (1%). The concentration of protein was measured with a BCA Assay Kit (Thermo Scientific). Protein samples were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore). The membrane was blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4 °C in the diluted primary antibody solution. After incubation with horseradish peroxidase-coupled secondary antibody for 1 h (Dako, 1:5000), the bands were washed in TBST solution three times. Then the band signals were detected with enhanced chemiluminescence (ECL) (PerkinElmer) using the ImageQuant LAS 4000 system (GE Healthcare Life Sciences). The following antibodies were used: rabbit anti-HOXD10 (Abcam, Ab138508, 1:5000), rabbit anti-mTOR (Abcam, Ab32028, 1:2500), rabbit anti-AKT (Ab18785), rabbit anti-AKT (phospho T308) (Ab38449) and mouse anti-human β-actin antibody (CST, 3700, 1:1000 dilution).
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