Protein was isolated from cells using NP40 lysis buffer (0.5% NP-40 (US Biologicals; NP40S), 50mM Tris (pH 7.5), 150mM NaCl, 3mM MgCl2, 1X protease inhibitors (Roche; 0505489001)). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific; 23227). For western blot analysis, equal protein concentrations were loaded onto and separated in 12% (w/v) sodium dodecyl sulfate polyacrylamide gel (40% acrylamide/bis-acrylamide solution; Bio-Rad; 161-0146). Proteins were transferred from the gel to 0.45 mm pore size nitrocellulose membrane (Maine Manufacturing; 1215471) and total protein visualized using Ponceau S (Amresco; K793). The membrane was blocked with 2.5% (w/v) bovine serum albumin (BSA; Fisher; BP 1600-1) in 1X TBST (20mM Tris, pH 7.6, 150mM NaCl, 0.002% Tween-20). Primary and secondary antibodies were diluted in 2.5% BSA/1X TBST. Protein blot bands were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific; 34095) and imaged using the GE Amersham Imager 600 (General Electric). Primary antibodies: AR (Cell Signaling; D6F11), p62/SQSTM1 (Abnova; H00008878-M01), SOD2 (Abgent; AM7579a), β-actin (Abcam; ab8226), β-actin (Novus; NB600-505), NKX3.1 (Cell Signaling, D2Y1A). Secondary antibodies: Sheep anti-mouse (Jackson ImmunoResearch Laboratories; 515-035-062), goat anti-rabbit (Abnova; PAB10822).
Acrylamide bis acrylamide solution
Manufactured by Bio-Rad
Sourced in United States
Acrylamide/bis-acrylamide solution is a reagent used in gel electrophoresis. It is a mixture of acrylamide and N,N'-methylenebisacrylamide, which are polymerized to form the gel matrix. This solution is a key component in the preparation of polyacrylamide gels for the separation and analysis of proteins, nucleic acids, and other macromolecules.
Automatically generated - may contain errors
Lab products found in correlation
Ammonium persulfate, by Bio-Rad (3 mentions)
Temed, by Bio-Rad (3 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Ponceau s, by Avantor (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Amersham imager, by Cytiva (2 mentions)
Glutaraldehyde, by Merck Group (2 mentions)
Temed, by Merck Group (2 mentions)
P62 sqstm1, by Abnova (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
β actin, by Novus Biologicals (1 mentions)
β actin, by Abcam (1 mentions)
Ge amersham imager 600, by GE Healthcare (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Np 40, by US Biological (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Hydrogen peroxide h2o2, by Merck Group (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
14 protocols using acrylamide bis acrylamide solution
1
Western Blot Protein Quantification
2
Protein Isolation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was isolated from cells using NP40 lysis buffer (0.5% NP40 [US Biological, Salem, MA; N3500], 50 mM of Tris [pH 7.5], 150 mM of NaCl, 3 mM of MgCl2, 1X protease inhibitors [Roche, Mannheim, Germany; 05892953001]). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA; 23227). For Western blot analysis, equal protein concentrations were loaded onto and separated in 12% (wt/vol) sodium dodecyl sulfate polyacrylamide gel (40% acrylamide/bisacrylamide solution; Bio‐Rad, Hercules, CA; 161‐0148). Proteins were transferred from the gel to 0.45 μm pore size nitrocellulose membrane (Maine Manufacturing, Sanford, ME; 1215471) and total protein visualized using Ponceau S (Amresco, Radnor, PA; K793). The membrane was blocked with 2.5% (wt/vol) BSA (Thermo Fisher Scientific, Waltham, MA; BP 1600‐1) in 1X tris-buffered saline with Tween 20 (TBST; 20 mM of Tris, pH 7.6, 150 mM of NaCl, 0.05% Tween‐20). Primary and secondary antibodies were diluted in 2.5% BSA in 1X TBST. Protein blot bands were visualized using Clarity Western ECL Substrate (Bio‐Rad, Hercules, CA; 1705061) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Rockford, IL; 34095) and imaged using Amersham Imager 600 (GE, Marlborough, MA).
3
Differential Alkylation Proteomics Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All reagents used in cell culture were cell culture-tested. The Dulbecco's modified Eagle medium (DMEM) with Glutamax™ and low glucose (1 g/L), fetal bovine serum (FBS), trypsin 0.05% solution in phosphate buffered saline (PBS), Fungizone® Antimycotic (amphotericin B), penicillin-streptomycin solution (Pen-Strep) solution, and Dulbecco′s phosphate buffered saline (DPBS) (10×) were obtained from Invitrogen. The hydrogen peroxide (H2O2) used in oxidative stress stimulation was obtained from Sigma-Aldrich.
The reagents used in the differential alkylation procedure have different origins: the iodoacetamide was obtained from Sigma-Aldrich, the 40% acrylamide/bis-acrylamide solution (37.5:1) from BioRad and the dithiothreitol (DTT) from GE Healthcare Life Sciences. For in gel digestion, the precast polyacrylamide gels “4–20% TGX Stain-Free Gel” and all the buffers, including the Laemmli buffer, used in the electrophoresis were obtained from Bio-Rad, and the Trypsin Modified Sequencing Grade used in protein digestion, was obtained from Roche Diagnostics.
The reagents used in MS analysis were all high-quality chemical or reagents (ACS Reagent Chemicals & Lab Grades). Formic acid (FA) was obtained from AMRESCO and water, methanol and acetonitrile (ACN) from Fisher. Ortho-phosphoric acid and ammonium sulfate were from MERK and ammonium bicarbonate from Fluka.
The reagents used in the differential alkylation procedure have different origins: the iodoacetamide was obtained from Sigma-Aldrich, the 40% acrylamide/bis-acrylamide solution (37.5:1) from BioRad and the dithiothreitol (DTT) from GE Healthcare Life Sciences. For in gel digestion, the precast polyacrylamide gels “4–20% TGX Stain-Free Gel” and all the buffers, including the Laemmli buffer, used in the electrophoresis were obtained from Bio-Rad, and the Trypsin Modified Sequencing Grade used in protein digestion, was obtained from Roche Diagnostics.
The reagents used in MS analysis were all high-quality chemical or reagents (ACS Reagent Chemicals & Lab Grades). Formic acid (FA) was obtained from AMRESCO and water, methanol and acetonitrile (ACN) from Fisher. Ortho-phosphoric acid and ammonium sulfate were from MERK and ammonium bicarbonate from Fluka.
4
Recombinant Human Polymerases for Gel Electrophoresis
5
Polyacrylamide Gel Preparation for Cell Mechanics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fibronectin-coated PAA gels containing 0.2 μm fluorescent microspheres (Life Technologies) were prepared on glass-bottomed dishes, as described previously (Pelham and Wang, 1997 (link)). In brief, the glass surfaces were incubated with 0.1 N NaOH and air-dried. The surfaces were then incubated with 3-aminopropyltrimethoxysilane (Sigma) and 0.5% glutaraldehyde (Sigma), and washed with distilled H2O in between incubations. After drying, a drop of acrylamide/bis-acrylamide solution containing ammonium persulfate (BioRad, Hercules, CA), tetramethylethylenediamine (TEMED; Sigma), and 0.2 μm fluorescent microspheres was pipetted onto the modified glass surface. A coverslip was placed over the droplets to ensure a flat gel surface after polymerization. 10 µg/ml of fibronectin was coupled to the PAA substrates via the bi-functional crosslinker sulfosuccinimidyl hexanoate (sulfo-SANPAH; Pierce Biotechnology, Rockford, IL). Gels with elastic moduli of 8 kPa and 35 kPa were generated with acrylamide/bis-acrylamide ratios of 5%/0.3% and 10%/0.2%, respectively. The elastic moduli were measured using a rheometer (AR-G2; TA Instruments, New Castle, DE). To accommodate fluorescent imaging with GFP and mCherry markers in cells, dark red fluorescent beads (660/680; Life Technologies) were used, at a final concentration of 0.0032% by volume.
6
Western Blot Analysis of TET1 and TET3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates were prepared using AllPrep DNA/RNA/Protein Kit (Qiagen), according to the manufacturer’s instructions. Polyacrylamide gels were poured in 8 % concentration using 30 % acrylamide/bisacrylamide solution (Bio-Rad), sodium dodecyl sulfate (SDS) (Bio-Rad), ammonium persulfate (APS) (Bio-Rad), tetramethylethylenediamine (TEMED) (Bio-Rad), Resolving Gel Buffer (Bio-Rad) and Stacking Gel Buffer (Bio-Rad). Electrophoresis of protein lysates was performed using 10 µL of each sample, followed by Ponceau S staining. Samples were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). The following antibodies were used for the detection of TET1, TET3, and beta-actin: anti-TET1 in dilution of 1:1000 (Thermo Fisher, PA5-72805), anti-TET3 in dilution 1:1000 (Abcam, ab 139311) and anti-beta-actin in dilution of 1:10 000 (Cell Signaling, 4967L). All membranes were incubated with a secondary anti-rabbit IgG antibody in a dilution of 1:7500, conjugated to horseradish peroxidise (HRP) (Thermo Fisher). Protein signals were detected by chemiluminescence using Clarity Max Western ECL Substrate (Bio-Rad) and quantified with ImageLab software (Bio-Rad). TET1 and beta-actin measurements were normalized to whole-cell lysate content based on the Ponceau S staining.
7
DNA Size Separation by Polyacrylamide Gel Electrophoresis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A vertical polyacrylamide gel electrophoresis setup (Bio-Rad Protean II, Gel Company Inc., San Francisco, CA, USA) was used for DNA size separation. ssDNA oligomers were diluted in gel loading solution (1% glycerol), and were run on a 20% non-denaturing polyacrylamide gel (1× TAE buffer and Acrylamide/Bis-Acrylamide solution 19:1, BIO-RAD, Hercules, CA, USA), for 4 h at 90 V. The gel was stained for 15 min in 1× GelRed (Merck, Darmstadt, Germany) and was visualized on a transilluminator.
8
Sperm DNA Fragmentation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DSB DFI was performed by LensHooke® R11 Sperm DNA Fragmentation Releasing Assay (Bonraybio). A semen aliquot of 70 μL was mixed with 70 μL 30% (w/v) acrylamide/bis-acrylamide solution (BioRad, Hercules, CA), 15 μL 1% (w/v) ammonium persulfate (BioRad), and 15 μL tetramethylethylenediamine (TEMED, BioRad) to initiate gel polymerization in a 1.5 mL microcentrifuge. A 15 μL aliquot of the mixture was immediately placed onto the pretreated microscope slide and covered by a 24 × 40 mm coverslip. The slide was horizontally placed at RT for 5 min and the coverslip was carefully removed. The lysis solution (0.4 M Tris, 1 M Urea, 0.05% SDS, 50 mM TCEP, 50 mM Na2EDTA, 2.5 M NaCl, 1% Triton X-100, and 5 mM NaOH, pH 8.0) was immediately added to the slide and the slide was incubated at RT for 10 min, followed by tilting to drain off the residual reagents. The slide was immersed in distilled water for 5 min, Diff-Quik I for 1 min, Diff Quik II for 1 min, and de-stained with 75% ethanol for 1 min. The dried slide was evaluated under a bright-field microscope. Sperm with a large and/or medium halo were referred to as sperm with DSBs. DSB DFI was calculated as the percentage of spermatozoa with a halo over the total population. A minimum of 500 spermatozoa were scored per test sample.
9
Cell Culture and Western Blot Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RPMI 1640 media and fetal bovine serum (S001-01) were purchased from Wellgene (Gyeongsan-si, Gyeongsangbukdo, Korea). Trypsin-Versene (EDTA) and antibiotics (penicillin–streptomycin) were from Lonza (Basel, Switzerland). Recombinant human EGF (PHG0311) was from Invitrogen (Carlsbad, CA, USA). Thiazolyl blue tetrazolium bromide for MTT assays was purchased from Sigma (St. Louis, MO, USA). Pro-prep lysis buffer for protein extraction was obtained from iNtRON Biotechnology (17081, Seongnam-si, Gyeonggi-do, Korea). Western blot reagents, including 30% acrylamide/bis-acrylamide solution, Tris–HCl (pH 6.8), Tris–HCl (pH 8.8), ammonium persulfate, and 10% sodium dodecyl sulfate (SDS), were from Bio-Rad (Hercules, CA, USA). Mouse anti-CD29 and cytosolic anti-integrin β1 antibodies were purchased from BD Biosciences (San Jose, CA, USA) and Abcam, (Cambridge, UK), respectively. Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, D16H11, rabbit), anti β-actin (13E5, rabbit), and anti Rab25 (D4P6P, rabbit) antibodies were from Cell Signaling Technology (Danvers, MA, USA). All antibodies were diluted 1:1000, except for those against GAPDH and β-actin, which were used at 1:2000. Anti-mouse and anti-rabbit IgG horse radish peroxidase (HRP)-conjugated secondary antibodies were manufactured by Santa Cruz Biotechnology (Dallas, TX, USA).
10
Quantitative Analysis of Seed Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lathyrus sativus seeds (chickling vetch, variety unknown) were kindly provided by Cover Crop Canada. Pisum sativum seeds: CDC Amarillo, CDC Meadow, CDC Limerick, CDC Inca, and CDC Dakota were generously provided by CDC (Crop Development Centre), University of Saskatchewan, commercial yellow pea (unknown cultivar), bovine serum albumin (BSA), horseradish peroxidase (HRP, Type I, 96 U/mg solid), putrescine (1,4-diaminobutane dihydrochloride), 4-amino-antipyrine (AAP), 3,5-dichloro-2-hydroxybenzenesulfonate sodium (DCHBS), β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), and glycyl-L-aspartic acid (Gly-Asp). Membranes of cellulose (12–14 kDa cut-off) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Bradford reagent and the acrylamide/bis-acrylamide solution (29:1) were purchased from Bio-Rad Laboratory (Mississauga, ON, Canada). All other chemicals were reagent-grade and were used without further purification.
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!