Whole brains were collected from male hKO and WT mice (n = 4 of each genotype) and mushed and lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors. Protein concentration in each sample lysate was adjusted to 0.5 μg/μL with 2× sample buffer. An equal amount volume of each lysate (10 μL) containing 5 µg protein was loaded onto Extra PAGE One precast gels (7.5%, Nacalai Tesque, Kyoto, Japan). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using WSE-7210 EzFastBlot HMW (Atto). The membrane was blocked with 5% ECL Prime blocking agent (GE) and incubated with primary antibody, anti-ARID1B (ab57461, abcam) or anti-TUBULIN (PM054, MBL), followed by secondary antibody, either anti-Mouse IgG HRP (#7076, CST) or anti-Rabbit IgG HRP (NA934VS, GE), respectively. The bands were visualized with ECL Prime (GE). Band intensities were quantified by densitometry using the ImageJ software and the quantity of ARID1B protein in each sample was normalized to that of TUBULIN.
Ab57461
Manufactured by Abcam
Sourced in United Kingdom
Ab57461 is a mouse monoclonal antibody that recognizes the human TPBG protein. It can be used for applications such as ELISA, Western Blot, and Immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ecl prime, by GE Healthcare (1 mentions)
Anti rabbit igg hrp, by GE Healthcare (1 mentions)
Extrapage one precast gels, by Nacalai Tesque (1 mentions)
Enhanced chemiluminescence detection kit, by Thermo Fisher Scientific (1 mentions)
Immun blot polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ab6728, by Abcam (1 mentions)
Ab230879, by Abcam (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
On targetplus, by Horizon Discovery (1 mentions)
Smarca4, by Santa Cruz Biotechnology (1 mentions)
Smarca2, by Santa Cruz Biotechnology (1 mentions)
Sc 25778, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
5 protocols using ab57461
1
ARID1B Protein Expression in Mice
2
Western Blot Analysis of ARID1B Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and tissues were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.2), 0.5% NP-40, 1% Triton X-100, 1% sodium deoxycholate) containing a protease inhibitor cocktail (Sigma-Aldrich Corporation, MO, USA). Cell and tissue lysates were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to an Immun-Blot® polyvinylidene fluoride membrane (Bio-Rad Laboratories, Inc. CA, USA). The membranes were blocked with 5% non-fat milk in TBST and subsequently probed with primary ARID1B antibodies (1:1000, ab57461; Abcam, Cambridge, UK) overnight at 4°C. Mouse anti-b-actin monoclonal antibody (1:3000; cat. no. 60008; Proteintech Group, Inc., IL, USA) was used as an internal control. Primary antibodies were detected by incubating the membranes with a horseradish peroxidase-conjugated secondary antibody (1:3000; ab6728; Abcam, Cambridge, UK) for 1 h at room temperature. The blots were subsequently developed using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Inc. IL, USA) and exposure to film.
3
Immunohistochemical Analysis of ARID1B and HOXA3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
4
Evaluating ARID1B Expression in Tissue Microarrays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue microarrays were constructed as described previously. Primary anti-ARID1B antibody (3 µg/ml, ab57461; Abcam, Cambridge, UK) was used for immunohistochemical staining. Two independent pathologists who were blinded to the clinicopathological data and outcome of each patient evaluated the staining intensity of the specimen. A semi-quantitative immunoreactivity scoring system was used for this evaluation as reported elsewhere. X-tile software (version 3.6.1, Yale University, New Haven, CT) was applied to select the optimum cutoff score for the staining intensity to separate patients into high and low ARID1B expression groups.
5
Targeted Silencing of Chromatin Remodelers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNAi knockdown of SMARCA4, SMARCA2, ARID1A and ARID1B was done by Lipofectamine 2000 (Invitrogen) transfection of Dharmacon ON-TARGETplus siRNA pools (20nM final), in comparison to Non-Targeting Control (NTC) siRNA pool. Previous studies demonstrated on-target knockdown, as two or more individual siRNAs from each pool exhibited similar knockdown efficiency and cellular phenotype [7 (link)]. Re-expression of SMARCA4 was done by retroviral transduction (pBABE-puro-SMARCA4) [7 (link)], in comparison to empty vector control. Knockdown and/or re-expression were confirmed by western blotting of whole cell lysates, using the following primary antibodies: SMARCA4 (sc-17796; Santa Cruz), SMARCA2 (sc-166579; Santa Cruz), ARID1A (ab176395; Abcam), ARID1B (ab57461; Abcam), and GAPDH (sc-25778; Santa Cruz). Knockdown efficiency was quantified using ImageJ.
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!