The cells were lysed in lysis buffer (Beyotime) and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel electrophoresis to separate proteins by molecular weight. Next, the proteins were blotted onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Then, the membranes were treated with 5% non-fat milk for 1 h. Next, the membranes were incubated with primary antibodies against Cyclin D1 (ab16663; 1/100; Abcam, Cambridge, MA, USA), CDK4 (ab108357; 1/5000; Abcam), Bcl-2 (ab32124; 1/1000; Abcam), Bax (ab32503; 1/4000; Abcam), MMP2 (ab37150; 1 µg/mL; Abcam), MMP9 (ab38898; 1/1000; Abcam), KLF6 (ab241385; 1/1000; Abcam) and GAPDH (ab8245; 1/2000; Abcam) overnight at 4°C. Next, the membranes were interacted with horseradish peroxidase-conjugated secondary antibody (ab6721; Abcam) with 1/3000 dilution at room temperature for 2 h. Finally, the protein bands were visualized by enhanced chemiluminescence chromogenic substrate (Beyotime) and measured by Image Lab software (Bio-Rad).
Ab241385
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab241385 is a laboratory equipment product. It is a core component used in various scientific research and analysis procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ab8245, by Abcam (2 mentions)
Image lab software, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab37150, by Abcam (1 mentions)
Enhanced chemiluminescence chromogenic substrate, by Beyotime (1 mentions)
Ab108357, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab38898, by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
Ab97051, by Abcam (1 mentions)
Ecl detection system, by Merck Group (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Pvdf membrane, by Abcam (1 mentions)
Ab245308, by Abcam (1 mentions)
Ab109520, by Abcam (1 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Minute protein extraction kit, by Invent Biotechnologies (1 mentions)
4 protocols using ab241385
1
Western Blot Analysis of Signaling Proteins
2
Immunohistochemical Analysis of KLF6 and SGMS2 in LUSC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We collected nine pairs of LUSC tumor tissue and adjacent normal tissue samples from January, 2016 to January, 2017 at the Sun Yat‐Sen university cancer center. The FFPE tissue sections were dewaxed and rehydrated, and subjected to antigen retrieval and blocking. First of all, the slides were incubated with anti‐rabbit primary antibodies overnight at 4°C. The primary antibodies included anti‐KLF6 polyclonal antibody (1:1000; ab241385; ABCAM) and anti‐SGMS2 polyclonal antibody (1:1000; ab237681; ABCAM). Then, after incubation with HRP‐conjugated rabbit polymer (1:500; ab97051; ABCAM), the secondary antibody, we used liquid diaminobenzidine tetrahydrochloride, and substrate (DAB chromogen, Changjiang) followed by counterstaining with hematoxylin for visualization. Finally, a light microscope (Nikon) was used to photograph the samples, which were analyzed using ImageJ (FIJI v2.1.0). A semiquantitative scoring system was conducted to assess and score the sample ranging from 1 (minimum) to 4 (maximum).
3
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein extraction of cell lines was conducted applying RIPA buffer (Pierce 89,900) according to the manufacturer's instructions. The proteins were separated on a 10% SDS-PAGE, which were then transferred onto PVDF membrane (GE Healthcare, NJ, USA). Next, the PVDF membrane was incubated with primary antibodies (GAPDH, 1:5000, ab8245, Abcam; KLF6, 1:1000, ab241385, Abcam; p21, 1:2000, ab109520, Abcam; E2F1, 1:4000, ab245308, Abcam; HIF1A, 1:1000, 20960-1-AP, Proteintech) at 4°C all through the night. Then, the membranes were washed in TBST buffer for 3 times and incubated with specific secondary antibody for 1 h. Finally, the membranes were visualized via an enhanced chemiluminescence (ECL) detection system (Millipore, USA).
4
Protein Extraction and Immunoblotting Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysis buffer (Minute™ Protein Extraction Kits, Invent, MN, USA) was applied for cell collection. BCA protein assay (KGP902, KeyGEN BioTECH, Jiangsu, China) was operated to quantify protein concentration. Identical quantities of protein (30 μg/lane) were exposed to 12% SDS-PAGE before transferring to polyvinylidene difluoride (PVDF, Millipore, Bedford, MA, USA) membranes. Following being blocked in 5% fat-free dry milk, anti-cleaved caspase-3 antibodies (1 : 1000, Cell Signaling Tech, Danvers, MA, USA) or anti-KLF6 antibody (1 : 1000, ab241385, Abcam, Cambridge, MA, UK) was applied to the membranes for incubation at 4°C overnight. Anti-GAPDH (1 : 5000, AP0066, Bioworld Tech, MN, USA) was used as an internal control. Following being washed, horseradish peroxidase-linked goat anti-rabbit IgG (1 : 3000, BS13278, Bioworld Tech, MN, USA) was applied to incubate the membranes for 1 h. Bound antibodies were identified with the implementation of the ECL detection system (Carestream, NY, USA), and the immunoreactive bands were quantified utilizing ImageJ 1.41o software (National Institute of Mental Health, 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!