Proteins for Western blotting were isolated using TIVE lysis buffer (50 mM Tris-HCl pH 7.8, 2 mM EDTA, 150 mM NaCl, 1% NP-40, protease inhibitors), or lysis buffer A (10 mM HEPES pH 7.5, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, protease inhibitors) plus 0.5% NP-40 and lysis buffer C (20 mM HEPES pH 7.5, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% Glycerol, 1 mM DTT, protease inhibitors) for total, cytoplasmic and nuclear proteins respectively. Co-immunoprecipitation (co-IP) was carried using the nuclear protein fraction, similarly to previously described (Jehle et al., 2014 (link)) and ARv7 antibody-coupled Protein A Dynabeads (Thermo Fisher Scientific). Co-IP experiments with formaldehyde crosslinked material was carried out using isolated chromatin samples, as described below. Western blotting was carried out using following antibodies: AR (N20 and 441, Santa-Cruz Biotechnologies; PG21, Millipore), AR C-terminus (SP242, Spring Bioscience; C-19, Santa-Cruz Biotechnologies), ARv7 (RM7, RevMAb; H6 253, Epitomics), NCOR1 (PA1–844A, Invitrogen), NCOR2 (ab24551, Abcam), FOXA1 (ab5089, Abcam), b-Actin (Abcam) and Lamin B1 (EPR8985, Abcam), and secondary antibody IgG fraction monoclonal mouse anti-rabbit IgG, light chain specific (211–032-171, Jackson ImmunoResearch).
Pg 21
Manufactured by Merck Group
Sourced in United States
The PG-21 is a laboratory equipment product manufactured by Merck Group. It is a device designed for the precise measurement and analysis of various physical and chemical parameters in a controlled laboratory environment. The core function of the PG-21 is to provide accurate and reliable data to support scientific research and testing activities.
Automatically generated - may contain errors
Lab products found in correlation
Ar441, by Santa Cruz Biotechnology (2 mentions)
Ab5188, by Abcam (2 mentions)
Phospho akt ser473, by GeneTex (2 mentions)
Phospho ser2448 mtor, by GeneTex (2 mentions)
Gapdh, by GeneTex (2 mentions)
Ab24551, by Abcam (1 mentions)
Ab5089, by Abcam (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Pa1 844a, by Thermo Fisher Scientific (1 mentions)
Anti pparγ antibody, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Bcrt102, by OriGene (1 mentions)
C18 column, by Waters Corporation (1 mentions)
Trypsin, by Promega (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
H 300, by Santa Cruz Biotechnology (1 mentions)
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (1 mentions)
17 protocols using pg 21
1
Protein Isolation and Co-immunoprecipitation Protocols
2
Androgen Receptor and PPARγ Regulation
3
Comprehensive Breast Cancer Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
AR antibody, PG-21, was obtained from Millipore (Billerica, MA). Actin antibody was procured from Chemicon International (Temecula, CA). Platypus migration assay was obtained from Fisher Scientific (Pittsburg, PA) and transwell migration chambers were obtained from Life Technologies (Carlsbad, CA). Breast cancer cDNA array, BCRT102, was obtained from Origene (Rockville, MD). All reagents used in the study were of analytical grade.
4
AR Interactome Profiling in LNCaP Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The LNCaP cells were treated
with drugs for 24 h and lysed in TBS, 0.1% Triton X-100, protease,
and phosphatase inhibitors (Roche). Immunoprecipitation was performed
using anti-AR (AR441, Santa Cruz). Western blot was used to detect
AR (PG-21, Millipore), RNA polymerase II (clone 8WG16; Covance), DDX5
(Millipore #05-850), or DDX17 (Millipore). The immunoprecipitated
AR was separated by SDS-PAGE and stained with SimplyBlue (Themo Fisher).
Gel bands were excised and destained in ammonium bicarbonate. After
disulfide bond reduction with 10 mM tris(carboxyethyl)phosphine and
thiol alkylation with 50 mM iodoacetamide, the gel bands were incubated
with trypsin (Promega) overnight. Peptides were extracted with 0.1%
trifluoroacetic acid/70% acetonitrile and lyophilized. The samples
were prepared and loaded along with a protein standard (yeast alcohol
dehydrogenase) for analysis on a MALDI Q-TOF instrument (Waters) following
separation by liquid chromatography using a C18 column (Waters). Peptide
analysis was performed using Scaffold (Proteome Software, Inc.), with
the relative quantities determined only on proteins with >95% probability
of identity and a minimum of five peptides per sample.
with drugs for 24 h and lysed in TBS, 0.1% Triton X-100, protease,
and phosphatase inhibitors (Roche). Immunoprecipitation was performed
using anti-AR (AR441, Santa Cruz). Western blot was used to detect
AR (PG-21, Millipore), RNA polymerase II (clone 8WG16; Covance), DDX5
(Millipore #05-850), or DDX17 (Millipore). The immunoprecipitated
AR was separated by SDS-PAGE and stained with SimplyBlue (Themo Fisher).
Gel bands were excised and destained in ammonium bicarbonate. After
disulfide bond reduction with 10 mM tris(carboxyethyl)phosphine and
thiol alkylation with 50 mM iodoacetamide, the gel bands were incubated
with trypsin (Promega) overnight. Peptides were extracted with 0.1%
trifluoroacetic acid/70% acetonitrile and lyophilized. The samples
were prepared and loaded along with a protein standard (yeast alcohol
dehydrogenase) for analysis on a MALDI Q-TOF instrument (Waters) following
separation by liquid chromatography using a C18 column (Waters). Peptide
analysis was performed using Scaffold (Proteome Software, Inc.), with
the relative quantities determined only on proteins with >95% probability
of identity and a minimum of five peptides per sample.
5
Quantitative Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 80 µg of protein was loaded into the wells of an 8–10% SDS-PAGE gel and subsequently transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Bucks, UK). Following this, the membranes were blocked with 5% skimmed milk for 1 h at room temperature. Next, the membranes were incubated overnight at 4 °C with primary antibodies against AR (1:500, PG-21, Millipore, Billerica, MA, USA) and CDYL (1:1000, ab5188, Abcam, Cambridge, UK). The protein bands were then visualized using an ECL kit (Millipore, Billerica, MA, USA). For Western blot densitometry and band quantification, image analysis software (Quantity One, Bio-Rad, Hercules, CA, USA) was utilized. GAPDH levels were employed as an internal control. This software allowed for the quantification of protein levels, including the amount of protein used, primary and secondary antibody binding, and subsequent detection. Loading control measures were employed to ensure accurate quantification.
6
Chromatin Immunoprecipitation Assay for CDYL and TNP1 Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
6 × 106 cells/mL were cross-linked in situ using 1% formaldehyde from Sigma-Aldrich (Burlington, MA, USA). Following cross-linking, the cells were lysed and subjected to sonication to fragment DNA into pieces ranging from approximately 300 to 1000 base pairs in size. The ChIP assay was carried out using the ChIP Assay Kit with Magnetic Protein A/G Beads, following the manufacturer’s recommendations provided by Millipore (Billerica, MA, USA). The pre-cleared chromatin was then subjected to immunoprecipitation with antibodies specific to AR (PG-21; Millipore, Billerica, MA, USA) or CDYL (ab5188, Abcam, Cambridge, UK) overnight. DNA fragments that formed complexes with the target protein were isolated and subsequently identified through quantitative PCR. This PCR was performed to validate the presence of the CDYL and TNP1 genes, with each sample undergoing triple repeats. The PCR cycles consisted of an initial step at 95 °C for 20 s, followed by 40 cycles of denaturation at 95 °C for 3 s and annealing/extension at 60 °C for 30 s. The PCR primers are: 5′-TCCATTCACAGGACGCTCAC-3′ and 5′-AGTGGGTGGCTTGACATCAG-3′ for CDYL, and 5′-CCTGACACTGTCCGTTCTCA-3′ and 5′-TGTGCATGGACCCTTTAGCC-3′ for TNP1.
7
Chromatin Immunoprecipitation Assay of AR, Gli Transcription Factors
8
Immunofluorescence Analysis of Androgen Receptor
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded on coverslips, fixed, permeabilized, blocked, and visualized (anti-AR; RRID:AB_310214; #PG-21, Millipore) and Alexa Fluor 568 (RRID:AB_10563566; #A11036; Molecular Probes) antibodies. Nuclei were visualized using ProLong Gold Antifade with DAPI (#P36941; Thermo Scientific). Cells were viewed under a Nikon Eclipse Ti-S fluorescent microscope and images captured using NIS-Elements software.
9
Androgen Receptor Localization in Cells
10
Western Blot Analysis of Signaling Proteins
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!