Total proteins of cell samples were extracted with lysis buffer and then quantified using the BCA method (KeyGen Biotech, Jiangsu, China). The lysate was diluted in SDS sample buffer (KeyGen Biotech) for SDS-polyacrylamide gelelectrophoresis (PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Roche Applied Sciences,USA). The membranes were immunoblotted at 4 °C overnight with anti-PRDX2 (Proteintech, USA), anti-c-Myc(Abcam, UK), anti-p-c-Myc(S62) (Abcam), anti-p-c-Myc(T58)(Abcam), anti-E-cadherin (Proteintech), anti-Vimentin (Proteintech), anti-N-cadherin (Proteintech), anti-GSK3β (Abcam), anti-p-GSK3β (Ser9) (Abcam), anti-AKT(1/2) (Abcam), anti-AKT1 (Abcam), anti-AKT2 (Abcam), anti-p-AKT2 (Ser474) (Abcam) and anti-GAPDH (Proteintech) antibodies at appropriate dilution concentration, followed by incubation using the appropriate second antibodies for 2 h. The bands were exposed using Pierce ECL Western Blotting Substrate (Thermo Scientific). Image J software was used to analyze the grey value of the interest protein.
Anti akt1
Manufactured by Abcam
Sourced in United Kingdom
Anti-AKT1 is a primary antibody that binds to the AKT1 protein. AKT1 is a serine/threonine protein kinase that plays a key role in cellular processes such as glucose metabolism, cell proliferation, and cell survival. The Anti-AKT1 antibody can be used in various applications to detect and study the AKT1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt2, by Abcam (2 mentions)
Anti β actin, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti pcna, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Anti vimentin, by Proteintech (1 mentions)
Anti p gsk3β ser9, by Abcam (1 mentions)
Anti e cadherin, by Proteintech (1 mentions)
Anti n cadherin, by Proteintech (1 mentions)
Anti gsk3β, by Abcam (1 mentions)
Anti prdx2, by Proteintech (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Bca method, by Keygen Biotech (1 mentions)
Sds sample buffer, by Keygen Biotech (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Anti c myc, by Abcam (1 mentions)
Corn oil, by Fujifilm (1 mentions)
Carbon tetrachloride ccl4, by Fujifilm (1 mentions)
Ly294002, by Merck Group (1 mentions)
8 protocols using anti akt1
1
Protein Quantification and Western Blot Analysis
2
Investigating CHI3L1 and Fas Signaling in Liver Injury
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Carbon tetrachloride (CCl4) and corn oil were from FUJIFILM Wako Pure Chemicals (Osaka, Japan). LY294002 was from Sigma‐Aldrich (St. Louis, MO, USA). Recombinant mouse CHI3L1 (rCHI3L1) and recombinant mouse Fas ligand (rFasL) were from R&D Systems (Minneapolis, MN, USA). Anti‐Mouse F4/80 antigen was from Tonbo Bioscience (San Diego, CA, USA). Anti‐AKT1 (phosphorylated Akt [pAkt]), anti‐Fas, anti‐CHI3L1, and goat anti‐rat IgG and L (Alexa Fluor 594) antibodies were from Abcam (Cambridge, UK). Goat anti‐rabbit IgG and L (Alexa Fluor 488) and anti‐mouse IgG and L (Alexa Fluor 594) antibodies were from Thermo Fisher Scientific (Waltham, MA, USA). Anti‐F4/80 antibody was from Hycult Biotech (Uden, The Netherlands). Anti‐CD68 monoclonal antibody (KP‐1) was from Agilent Technologies (Santa Clara, CA, USA). Jo2 (purified NA/LE hamster anti‐mouse CD95 antibody) was from BD Biosciences (San Jose, CA, USA).
3
Proteomic Analysis of BMSC Proteins
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Proteins were extracted from more than 106 harvested BMSCs according to the kit protocol (KeyGen Biotech, China). [46 (link)] BMSCs were digested in a lysis buffer cocktail and centrifuged at 12000 g for 15 minutes at 4 °C to get the supernatant as the total protein. 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) was used for electrophoresis of protein (20 μg) with loading buffer and then blotted to a polyvinylidene fluoride (PVDF) membrane (Millipore Co., USA). Membranes were blocked with 5% skimmed milk in Tris-buffered saline tween 20, TBST, and then incubated overnight at 4°C with primary antibodies; anti-IHH, anti-P53, anti-P16, anti-PI3K, anti-p-PI3K, anti-Akt 1, anti-p-Akt 1, anti-NF-κB, anti-p-NF-κB-p65, anti-STAT3, anti-p-STAT3, anti-4EBP1, anti-p- 4EBP1, anti-p70S6K, anti-p-p70S6K and anti-β-actin (1:1000), all from Abcam, USA. The membranes were incubated for 1hour at RT with secondary antibody conjugated with horseradish peroxidase. Finally, TBST-washed membranes were treated with enhanced chemiluminescent (ECL) for detection (Bio-Rad, USA). Image Lab detection system (Bio-Rad, USA) was used for protein bands imaging and analysis.
4
Western Blot Analysis of Protein Expression
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The predicted target protein expression was detected by western blotting. Briefly, total proteins were extracted from tendon tissue using RIPA lysis buffer (Applied Biosystems, MA, USA). Then, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, MA, USA). The membranes were blocked with 5% skimmed milk and incubated with primary antibodies anti-EGFR, anti-TP53, anti-AKT1, anti-MYC, and anti-GAPDH (1 : 1,000, Abcam, UK) at 4°C overnight. After washing with PBS, the membranes were incubated with a secondary antibody (1 : 1,000, Abcam). Enhanced chemiluminescent reagents (Millipore, MA, USA) were used to detect the protein bands. GAPDH was used as an internal control.
5
Protein Extraction and Western Blot Analysis
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Extracts of total proteins were obtained from cells with the use of radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) with the supplementation of 1% PMSF (Roche, Basel, Switzerland). After the separation of equal quantity of protein samples by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) gels, the proteins were shifted to polyvinylidene fluoride (PVDF) membranes, which were later sealed with skim milk. Blots were subjected to 12-h incubation with primary antibodies and then 1-h incubation with secondary antibody. Electrochemiluminescence (ECL) kit (Tanon) was applied for the visualization of protein signals. GAPDH was the internal control. Primary antibodies were anti-E-cadherin, anti-N-cadherin, anti-AKT1, anti-PCNA, and anti-Ki67 from Abcam (Cambridge, UK).
6
Astragaloside IV Modulates Immune Response
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Astragaloside IV (Solarbio, Science&Technology, Beijing, China; cat:SA8640; HPLC ≥ 98%). Dulbecco’s Modified Eagle Medium (DMEM; cat:12320032), fetal bovine serum (FBS; cat:26170043), and penicillin–streptomycin (PS; cat:10378016) were purchased from Gibco (Waltham, MA, USA). Dimethyl sulfoxide (DMSO; cat:D4540) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Estradiol benzoate injection (Jinke, Sichuan, China); progesterone injection (Xianju, Zhejiang, China); mifepristone tablets (Resources Zizhu, Beijing, China). Anti-IDO1(cat: ab211017), anti-PI3KCA (cat: ab40776), anti-AKT1(cat: ab179463), anti-LC-3(cat: ab48394), anti-FOXO3A (cat: ab154786), anti-PCNA (cat: ab92552), and anti-βactin (cat:ab8226) antibodies were purchased from Abcam (Cambridge, UK). Anti-caspase-9 (cat:10380-1-AP), anti-FADD (cat:14906-1-AP), and anti-PTEN (cat:22034-1-AP) antibodies were obtained from Proteintech (Wuhan, China). PerCP/Cyanine5.5 anti-rat CD3 (cat: 201417), Alexa Fluor® 488 anti-rat CD4 (cat: 201511), and APC anti-rat CD25 (cat: 202113) were purchased from Biolegend (San Diego, CA, USA). FOXP3 Monoclonal Antibody (150D/E4)-PE (eBioscience, San Diego, CA, USA; cat:12477441).
7
Protein Expression Analysis in Rat Spinal Cord Post-SCI
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Rat spinal cord tissue (dissociated at 7dpi post-SCI) was lysed in the denaturing buffer of the Minute™ Total Protein Extraction Kit (Cat# SD-001, Invent Biotechnologies, Beijing, China) to obtain protein extracts. The protein concentration of the supernatant was detected using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) with a full-wavelength functional microplate reader (Infinite M200Pro, Tecan, Switzerland). Proteins (30 μg/sample) were separated using 12.5% SDS–PAGE and transferred to nitrocellulose membranes. After blocking in 10% nonfat dry milk for 1 h, phosphorylated proteins were blocked using bovine serum albumin (5% BSA, room temperature) for 2 h. The membranes were incubated overnight at 4 °C with the following primary antibodies: Bcl-2 (1:500, Affinity Biosciences); cleaved caspase-3 (Asp175) (1:1000, Cell Signaling Technology); BAX (1:6000, Proteintech); Akt1 (1:1000; Abcam ab233755); Anti-Akt1 (phosphor S473, 1:4000, Abcam, ab81283); β-Actin (1:6000; Signalway Antibody). Appropriate HRP-conjugated secondary antibodies (1:80,00, Abmart, Shanghai, China) were applied for 1 h at room temperature. After 3 PBS-T washes as described above three times, the membranes were visualized using the ECL Western Blot Detection Kit (Merck, USA). Analysis was performed using the Bole CHEMIDOCXRS chemiluminescence imaging system and QuantityOne software.
8
Western Blot Analysis of AKT Proteins
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Proteins were extracted from tissues or cell lysates using a protein extraction kit (Sigma, USA) or RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). BCA protein assay kit (Beyotime, Shanghai, China) was used to determine the protein concentration in each sample. Equal amounts of proteins were mixed with the loading buffer and subjected to SDS-PAGE, and the proteins were then transferred onto Hybond ECL membranes (Amersham, Buckinghamshire, UK). The membranes were blocked in 5% non-fat milk and incubated with primary antibodies (anti-AKT1, Novus or anti-AKT2, Abcam) at 4°C overnight. The membranes were probed with HRP-labeled secondary antibodies and the protein bands were visualized using an enhanced chemiluminescence system (Kodak, Rochester, NY, USA). Densitometric quantification for all bands was analyzed with Image Pro-plus 6.0 (Media Cybernetics, USA).
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