Proteins (15–20 μg/well) were isolated on 10% sodium dodecyl sulfate and polyacrylamide gels, and the obtained bands were electronically transferred onto polyvinylidene fluoride membranes, followed by blockage with Tris-buffered saline (TBS) and 5% skimmed milk at room temperature for 60 min. The membranes were incubated at 4°C with primary antibodies: anti-PTEN (1:1000, SAB1406331, Sigma), anti-phospho-Akt (1:500, SAB4504331, Sigma), anti-Akt (1:2000, SAB4500797, Sigma), and anti-Actin (1:5000, A2066, Sigma) antibodies. The membranes were washed twice with TBS, to which 0.1% Tween-20 was added prior to incubation with secondary antibodies. The Ag/Ab complex was examined using the enhanced chemiluminescence detection kit with β-actin as the loading control.
Sab4500797
Manufactured by Merck Group
Sourced in United States
The SAB4500797 is a laboratory equipment product. It is designed to perform specific functions in a laboratory setting. The core function of this product is to facilitate scientific research and analysis, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Sab4504331, by Merck Group (3 mentions)
A2066, by Merck Group (1 mentions)
Ab37647, by Abcam (1 mentions)
Ab154244, by Abcam (1 mentions)
Ab18206, by Abcam (1 mentions)
Ab121009, by Abcam (1 mentions)
Hp5002, by Hycult Biotech (1 mentions)
Sab1405848, by Merck Group (1 mentions)
Gel documentation system, by Bio-Rad (1 mentions)
St506, by Beyotime (1 mentions)
Ab46545, by Abcam (1 mentions)
Ab131088, by Abcam (1 mentions)
Ab80508, by Abcam (1 mentions)
Ab32199, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab32370, by Abcam (1 mentions)
Sab5500162, by Merck Group (1 mentions)
Dmso d2650, by Merck Group (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab211292, by Abcam (1 mentions)
7 protocols using sab4500797
1
Western Blot Analysis of PTEN and Akt
2
Oxidative Stress Signaling in Aorta and HUVEC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated thoracic aortic tissues or cultured HUVECs were lysed in commercially purchased RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) spiked with 1 mmol/L phenylmethanesulfonyl fluoride (ST506, Beyotime, Shanghai, China) to obtain protein products. The antibodies against the following proteins were used: PXDN (1 μg/mL, ABS1675, Merck, Frankfurter, Germany), RAGE (0.3 μg/mL, ab37647, Abcam, Cambridge, UK), NOX2 (0.25 μg/mL, ab80508, Abcam, Cambridge, UK), NOX1 (0.5 μg/mL, ab131088 and ab121009, Abcam, Cambridge, UK), NOX4 (0.33 μg/mL, ab154244, Abcam, Cambridge, UK), 3-chlorotyrosine (0.1 μg/mL, 3-Cl-Tyr, HP5002, Hycult biotech, Uden, Netherlands), 4-hydroxynonenal (0.43 μg/mL, 4-HNE, ab46545, Abcam, Cambridge, UK), Akt (0.5 μg/mL, SAB4500797, Sigma-Aldrich, USA), p-Akt (0.143 μg/mL, ab18206, Abcam, Cambridge, UK), eNOS (0.25 μg/mL, 61029, BD, Biosciences, NJ, USA) and p-eNOS (0.056 μg/mL dilution, Ser1177, MA5-14957, Invitrogen, CA, USA). Expression of the protein GAPDH (0.5 μg/mL, SAB1405848, Sigma-Aldrich, MO, USA) was used for data normalization. PVDF membranes were incubated with a horseradish peroxidase linked secondary antibody and bands were visualized using gel documentation system (Bio-Rad).
3
Exploring Baicalein's Effect on NSCLC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Non‐small‐cell lung cancer A549 and H460 cell lines and the normal human bronchial epithelial (NHBE) cell line were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences (Shanghai, China); baicalein (465119) and dimethyl sulphoxide (DMSO, D2650) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Cisplatin was purchased from the Qilu Pharmaceutical Company (Jinan, Shandong, China). Primary antibodies for Western blotting against to PTEN (ab32199), survivin (ab469), Bcl‐xL (ab32370) and β‐actin (ab8227) was purchased from Abcam (Cambridge, UK); antibodies for PI3K (SAB5500162), total Akt (t‐Akt, SAB4500797) and phosphor‐forms (p‐Akt, SAB4301414) were purchased from Sigma‐Aldrich; the secondary antibody (ab205718) for Western blotting was purchased from Abcam (Cambridge, UK).
4
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were quantified by BCA method. Protein (30 µg) was mixed with loading buffer and denatured, followed by electrophoresis and transmembrane to polyvinylidene difluoride membrane (Merck, Darmstadt, Germany). Membranes were blocked with 5% skim milk at room temperature for 2 hours. After that, primary antibodies including anti-Prx1 (ab211292, 1:1,000, Abcam, Cambridge, UK), anti-FOXO3 (SAB2107951, 1:1,000, Sigma-Aldrich, St. Louis, MO, USA), anti-PI3K (GW21071, 1:500, Sigma-Aldrich), anti-p-PI3K (#SAB1305578, 1:1,000, Sigma-Aldrich), anti-Akt (SAB4500797, 1:1,000, Sigma-Aldrich), anti-p-Akt (#9271, 1:1,000, Cell Signaling Technology Danvers, MA, USA), and anti-GAPDH (ab37168, 1:1,000, Abcam) were used to incubate with the corresponding overnight at 4°C. After washing, membranes were incubated with goat antirabbit LgG (H + L) secondary antibody (1:1,000, Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd, Beijing, China). After washing, ECL detection reagent (Sigma-Aldrich, USA) was added to detect the signal. Relative expression level of each protein was normalized to endogenous control GAPDH using Image J software (https://imagej.nih.gov/ij/ ).
5
Western Blot Analysis of Protein Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from cultured cells at 70%–80% confluence, or from tumor/ANT tissues, separated by SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes. After incubation with primary monoclonal rabbit antibodies at 4°C for 12 h at room temperature, membranes were washed three times with tris-buffered saline and Tween 20 TBST) solution and then incubated with horseradish peroxidase-labeled secondary antibody (anti-rabbit IgG, 1:4,000, Sigma-Aldrich Co., St Louis, MO, USA) for 3 h. Membranes were washed with TBST and detected proteins visualized using ECL prime Western blotting detection reagent (Amersham Biosciences, Uppsala, Sweden).19 (link),20 (link) Rabbit anti-PBX3 antibody (1:1,000, ab56239) was from Abcam (Cambridge, MA, US), rabbit anti-pAKT (Ser473) (1:500, SAB4504331) was from Sigma, rabbit anti-total AKT (1:1,000, SAB4500797) was from Sigma, and anti-GAPDH (1:1,500, ab70136) was from Abcam.
6
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with lysis buffer, and proteins were extracted using a protein extraction kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. The proteins were separated by electrophoresis in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were electrotransferred onto polyvinylidene difluoride membranes. The membranes were blocked for 2 h with 5% non-fat milk at room temperature. Then, the membranes were incubated with primary antibodies against RBM8A (1 : 1000, HPA018403, Sigma-Aldrich), mTOR (1 : 1000, SAB4501039, Sigma-Aldrich), p-mTOR (1 : 1000, SAB4301415, Sigma-Aldrich), p-AKT (1 : 1000, SAB4504331, Sigma-Aldrich), AKT (1 : 1000, SAB4500797, Sigma-Aldrich), and β-actin (1 : 5000, SAB2701711, Sigma-Aldrich) at 4°C overnight. The membranes were then incubated with HRP conjugated secondary antibody (1 : 5000, 12–348, Sigma-Aldrich) for 1 h at room temperature. The immunoblots were measured by chemiluminescence detection system and quantified by ImageLab software (Bio-Rad, Hercules, CA, USA).
7
Western Blot Analysis of PI3K/AKT Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After transfection for 24 hours, Hs 766T or SW1990 cells were lysed by Western cell lysis buffer (Beyotime, Haimen, People's Republic of China). An equal amount of protein samples was loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and blotted onto polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated with 5% skimmed milk for 1 hour at room temperature for blocking. Primary antibodies against p-AKT (07–1398; Sigma), AKT (SAB4500797; Sigma), p-PI3K (ab389562; Abcam, Cambridge, MA, USA), PI3K (ab32089; Abcam), SLC7A11 (SAB2500951; Sigma) and β-actin (A1978; Abcam) were incubated with the membrane at 4°C overnight. Afterwards, horseradish-peroxidase (HRP)-labeled secondary antibody (Sigma) was incubated with the membrane for 2 hours at room temperature. Immunochemical detection was conducted with the enhanced chemiluminescence (ECL) Detection System (GE Healthcare, Chicago, IL, 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!