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Bis acrylamide

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Bis-acrylamide is a crosslinking agent used in the preparation of polyacrylamide gels for electrophoresis. It is a key component in the formation of the gel matrix that allows for the separation and analysis of proteins, nucleic acids, and other macromolecules.

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Lab products found in correlation

Acrylamide, by Bio-Rad (80 mentions) Ammonium persulfate, by Bio-Rad (11 mentions) Temed, by Bio-Rad (8 mentions) Ammonium persulfate, by Merck Group (7 mentions) Saponin, by Merck Group (7 mentions) Hepes, by Merck Group (7 mentions) Sulfo sanpah, by Thermo Fisher Scientific (6 mentions) Temed, by Merck Group (6 mentions) Sodium dodecyl sulfate (sds), by Bio-Rad (6 mentions) N n n n tetramethylethylenediamine, by Bio-Rad (5 mentions) Tween 20, by Merck Group (5 mentions) Bovine serum albumin (bsa), by Merck Group (5 mentions) Sodium dodecyl sulfate (sds), by Merck Group (5 mentions) Tetramethylethylenediamine, by Bio-Rad (4 mentions) Glycine, by Merck Group (4 mentions) Triton x 100, by Merck Group (3 mentions) N n n n tetramethylethylenediamine temed, by Bio-Rad (3 mentions) Methylglyoxal, by Merck Group (3 mentions) Protean 2 xi cell, by Bio-Rad (3 mentions) Coomassie brilliant blue, by Bio-Rad (3 mentions)

106 protocols using bis acrylamide

1

Fabrication of FN-PA Hydrogels

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FN-PA hydrogels with varying bisacrylamide concentrations were fabricated on amino-silanized coverslips, as previously described (Brown et al., 2013 (link)). In brief, PA gel solutions were produced by combining acrylamide and bisacrylamide (Bio-Rad) to final concentrations of 8% acrylamide and 0.045%, 0.102%, 0.146%, or 0.239% bisacrylamide to obtain gels with final elastic moduli of 1.8 kPa, 6.7 kPa, 10.6 kPa, or 18.7 kPa, respectively. 35 µl of each solution was polymerized by the addition of 1% (vol/vol) ammonium persulfate (VWR) and 0.1% (vol/vol) N,N,N′,N′-tetramethylethylenediamine (Bio-Rad). Human plasma FN was purified from blood plasma and covalently attached to the surface using 0.2 mg/ml UV-activated heterobifunctional cross-linker sulfosuccinimidyl-6-(4′-azido-2′nitrophenyl-amino)hexanoate (Pierce Chemical). After overnight incubation with 20 µg/ml FN, gels were then washed and stored in PBS.
Fiore V.F., Strane P.W., Bryksin A.V., White E.S., Hagood J.S, & Barker T.H. (2015). Conformational coupling of integrin and Thy-1 regulates Fyn priming and fibroblast mechanotransduction. The Journal of Cell Biology, 211(1), 173-190.
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2

SDS-PAGE Protein Separation and Visualization

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P7 or mP7 (1 μg) was prepared in sample buffer (25 mM Tris-HCL, 2% SDS (w/v), 0.01% (w/v) bromophenol blue) and heated (5 min, 95 °C). If indicated, samples were reduced by addition of 25 mM DTT to the sample buffer. Peptides were separated on an SDS-PAGE gel (Running gel; 18% (Bis)acrylamide (BioRad), 1 M Tris-HCL, 0.1% SDS (w/v), 10% glycerol (v/v), 0.1% ammonium persulfate (APS) (w/v), 0.04% TEMED (v/v) (BioRad) and Stacking gel; 4% (Bis)acrylamide, 1 M Tris-HCL, 0.1% SDS (w/v), 0.1% APS (w/v), 0.04% TEMED (v/v)). Directly after running (20 min, 50 V) followed by 120 min, 50 mA) gels were fixed in 5% glutaraldehyde for 60 minutes. After extensive washing with MilliQ, proteins were stained-in-gel with Coomassie blue solution (50% (v/v) methanol, 7% acetic acid, 0.1% (w/v) Coomassie blue). Background staining was removed with multiple washing steps with destaining solution (50% (v/v) methanol, 7% acetic acid) until peptide bands were properly visible. Images were made using ChemiDocTM MP Universal hood III (BioRad) and analyzed with the corresponding ImageLabTM software (version 5.2.1.). Protein weight was calculated with: http://www.sciencegateway.org/tools/proteinmw.htm, presented in Table 1.
Scheenstra M.R., van den Belt M., Tjeerdsma-van Bokhoven J.L., Schneider V.A., Ordonez S.R., van Dijk A., Veldhuizen E.J, & Haagsman H.P. (2019). Cathelicidins PMAP-36, LL-37 and CATH-2 are similar peptides with different modes of action. Scientific Reports, 9, 4780.
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3

Fabrication and Functionalization of Polyacrylamide Microparticles

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Polyacrylamide MPs were fabricated using an inverse emulsion polymerization protocol as previously described, with minor modifications (Labriola et al., 2016 (link)). Mechanically distinct MPs were created with either 4% acrylamide: 0.05% bis-acrylamide (“soft”) or 8% acrylamide: 0.3% bis- acrylamide (“stiff’) solutions (Bio-Rad, Hercules, CA). MPs were serially filtered with 40, 30, 20, and 15 μm mesh filters to decrease size dispersity. A triphenylmethane-based highlighter dye (Sharpie) with emission at 647 nm wavelength was added for visualization. MPs were subsequently functionalized by UV-photoactivation of a heterobifunctional cross linker, sulfo-SANPAH (#13414, CovaChem), followed by overnight incubation at 4 °C in a 100 μg/mL solution of collagen type 1 (COL-1, #08–115, Lot #2373345, Millipore). Uncoated MPs were used as controls.
Shah M.K., Leary E.A, & Darling E.M. (2018). Integration of hyper-compliant microparticles into a 3D melanoma tumor model. Journal of biomechanics, 82, 46-53.
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4

Polyacrylamide Gel Fabrication and Collagen Coating

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Polyacrylamide gels were made from 40% acrylamide and 2% bis acrylamide (Bio-Rad, Hercules, CA) where varying ratios of acrylamide and bis acrylamide were used to create gels of known reproducible stiffness (18 ). Gels were cast on glutaraldehyde-modified coverglasses and polymerization was activated by 1% ammonium persulfate (Sigma). The PA gel surfaces were then conjugated to acrylic acid N-hydroxy-succinimide ester activated by 365nm UV exposure and Irgacure catalyst (BASF Resins, Laramie, WY). Rat-tail collagen I (VWR, Brisbane, CA) was then added to the surface at 150ug/mL to allow coupling to the gel through side-chain primary amines.
Please refer to Supplemental Methods for additional methodological details.
Desai S.S., Tung J.C., Zhou V.X., Grenert J.P., Malato Y., Rezvani M., Español-Suñer R., Willenbring H., Weaver V.M, & Chang T.T. (2016). Physiological Ranges of Matrix Rigidity Modulate Primary Mouse Hepatocyte Function In Part Through Hepatocyte Nuclear Factor 4 Alpha. Hepatology (Baltimore, Md.), 64(1), 261-275.
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5

Polyacrylamide Gel Preparation

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40% acrylamide and 2% bis-acrylamide solutions (Bio-Rad, Hercules, CA, USA) were mixed with ddH2O to yield a 7.5% acrylamide and 0.01% bis-acrylamide solution. Polymerization was initiated by adding ammonium persulfate (APS) and tetramethylethylenediamine (TEMED). Gels were polymerized at room temperature in a well, overlaid with ddH2O, and cut into shape with a circular punch prior to measurements.
van Oosten A.S., Vahabi M., Licup A.J., Sharma A., Galie P.A., MacKintosh F.C, & Janmey P.A. (2016). Uncoupling shear and uniaxial elastic moduli of semiflexible biopolymer networks: compression-softening and stretch-stiffening. Scientific Reports, 6, 19270.
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6

Modulation of Extracellular Matrix Stiffness

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Poly-acrylamide (PA) gels of varying bisacrylamide concentrations were created on amino-silanated coverslips as previously described [68] . PA gel solutions were produced by combining acrylamide and bisacrylamide to final concentrations of 8% acrylamide (Biorad, Hercules, CA, USA) and 0.048%, 0.117%, 0.208%, or 0.260% bisacrylamide (Biorad) to obtain gels with final elastic moduli of 2 kPa, 8 kPa, 16 kPa, or 24 kPa, respectively. Fifty (50) µl of each solution was polymerized by the addition of ammonium persulfate (VWR, West Chester, PA, USA) and N,N,N′,N′-tetramethylethylenediamine (Biorad, Hercules, CA, USA) (1% and 0.1% final concentration respectively). The gels were allowed to polymerize for approximately 30 minutes, then washed three times with PBS. Fn was covalently attached to the surface using the heterobifunctional crosslinker sulfosuccinimidyl-6- (4′-azido-2′ nitrophenyl-amino) hexanoate (sulfo-SANPAH; Pierce Chemical Co., Rockford, IL., USA). Following an overnight incubation with the Fn, gels were washed three times with PBS.
Dysart M.M., Galvis B.R., Russell A.G, & Barker T.H. (2014). Environmental Particulate (PM2.5) Augments Stiffness-Induced Alveolar Epithelial Cell Mechanoactivation of Transforming Growth Factor Beta. PLoS ONE, 9(9), e106821.
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7

Polyacrylamide Gel Fabrication

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Soft gel mixes contained: 550 µl of 7.6 mM hydrochloric acid (HCL), 330.5 µl of double-distilled water (ddH2O), 0.5 µl N,N,N′,N′-tetramethylethylenediamine (TEMED) (Sigma), 20 µl 2% bis-acrylamide (BioRad), 70 µl of 40% acrylamide (BioRad), 20 µl 0.1 M NHS (N-hydroxysuccinimide, Sigma-Aldrich), 4 µl of 200 nm diameter beads resuspended at 0.2 µM (Invitrogen) and 5 µl of 10% ammonium persulfate (GE HealthCare) (prepared just before use). Stiff gels mixes contained: 550 µl of 7.6 mM HCL, 258.5 µl of ddH2O, 0.5 µl of TEMED (Sigma), 25 µl 2% bis-acrylamide (BioRad), 137 µl of 40% acrylamide (BioRad), 20 µl of 0.1 M NHS (N-hydroxysuccinimide, Sigma-Aldrich), 4 µl of 200 nm diameter beads resuspended at 0.2 µM (Invitrogen) and 5 µl of 10% ammonium persulfate (GE HealthCare) (added just before use). A 12-μl drop of PAA mix was placed into the hydrophilic glass of a glass bottom dish (FD5040-100). The PAA mix was covered with a hydrophobic 13-mm diameter × 0.1 mm glass coverslips that were prepared fresh by coating them with PlusONE Repel-Silene ES (GE Healthcare) for 15 min at room temperature and dried with an air pistol. Polymerization proceeded for 45 min at room temperature in a humidifier chamber. The coverslip was carefully removed, and gels were washed three times for 2 min with 10 mM HEPES.
Marchant C.L., Malmi-Kakkada A.N., Espina J.A, & Barriga E.H. (2022). Cell clusters softening triggers collective cell migration in vivo. Nature Materials, 21(11), 1314-1323.
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8

Homemade Laemmli Gel Electrophoresis

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The home-made Laemmli resolving gel (10 ml) contains 3 mL of 40% Acrylamide/Bis (cat#: 1610148, Bio-Rad), 2.5 mL of 1.5M Tris-HCl pH 8.8 (cat#: 1610798, Bio-Rad), 1 mL of 10% SDS (cat#: 1610416, Bio-Rad), 50 μl of 10% APS (cat#: 1610700, Bio-Rad) and 5 μl of TEMED (cat#: 1610800, Bio-Rad). The stacking gel (2.5 ml) contains 0.25 mL of 40% Acrylamide/ Bis, 0.63 mL of 0.5M Tris-HCl pH 6.8 (cat#: 1610799, Bio-Rad), 250 μl of 10% SDS, 12.5 μl of 10% APS and 2.5 μl of TEMED. The running buffer (1L) contains 3g Tris base (cat#: 11814273001, Sigma-Aldrich), 14.4g Glycine (cat#: G8898, Sigma-Aldrich), and 1g SDS (cat#: L3771, Sigma-Aldrich).
Wu Z., Tantray I., Lim J., Chen S., Li Y., Davis Z., Sitron C., Dong J., Gispert S., Auburger G., Brandman O., Bi X., Snyder M, & Lu B. (2019). MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure. Molecular cell, 75(4), 835-848.e8.
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9

Quantifying Cell Traction Forces

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To measure cell traction forces exerted by the cell, 5 kPa polyacrylamide (PAA) hydrogel substrates were prepared as described previously [44 ,45 ]. Briefly, 8% acrylamide and 0.1% bis-acrylamide (Bio-Rad), 2% N-Hydroxysuccinimide (dissolved in DMSO) (Sigma), 0.375 % 3-aminopropylsilyl (Thermo Fisher Scientific), 0.125% tetramethylethylenediamine (Millipore Sigma) were added in water to prepare the gels. In addition, 1% of 200 nm fluorescently labeled green beads (2% solid, Thermo Fisher Scientific) was added before gelation and the solution was left for 30 minutes to 1 h for polymerization at room temperature (RT). Sulfo-SANPAH was used for gel surface activation to facilitate protein conjugation for 3 – 5 min under UV illumination. Gels were laminated with rat tail collagen I at a concentration of 100 μg/mL. After 24 h of seeding, phase images, and fluorescence images of the bead were acquired before and after removal of the cells to relieve traction stress. For the TFM analysis, a custom-built Matlab code was used. The details of the calculation can be found in refs [44 ,46 (link)]. Briefly, the displacement field is calculated from stressed and relaxed images of the beads. A 32×32 pixel ROI is used for the image correlation calculation. By constrained Fourier transform traction microscopy (FTTM) the cell traction field is obtained from the displacement field.
Patteson A.E., Pogoda K., Byfield F.J., Mandal K., Ostrowska-Podhorodecka Z., Charrier E.E., Galie P.A., Deptuła P., Bucki R., McCulloch C.A, & Janmey P.A. (2019). Loss of vimentin enhances cell motility through small confining spaces. Small (Weinheim an der Bergstrasse, Germany), 15(50), e1903180.
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10

Capillary and Slab Gel Electrophoresis

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General procedure for preparing gel columns was followed. Briefly, 12% resolving gel was prepared by mixing 3.33 ml water, 2.5 mL Tris-HCl buffer (pH-8.8), 4 mL mixture of acrylamide and Bis-acrylamide (Bio-Rad) in ratio 29:1, 100 µL of 10% SDS, 60 µL Ammonium persulphate (APS) (Sigma Aldrich) and 6.5 µL of Tetramethylethylenediamine (TEMED) (Fisher Scientific). 6% stacking gel was prepared by adding 700 µL mixture of acrylamide: Bis-acrylamide in ratio 29:1, 675 µL of Tris-HCl buffer (pH-6.8) and 50 µL of 10% SDS. To this 25 µL of APS and 20 µL of TEMED were added. For capillary (Fisherbrand) preparation, resolving gel columns of 1.5 or 2.5 cm length were made by injecting 30 or 50 µL of resolving gel solution into the capillary tube, respectively. This was followed by injecting 10 µL of stacking gel solution to form stacking gel of 0.5 cm length. The standard PAGE slab was prepared using the above mentioned solutions to form resolving and stacking gels of 4 cm and 1 cm length.
Chaudhari S., Chaudhari K., Kim S., Khan F., Lee J, & Thundat T. (2017). Electrophoresis assisted time-of-flow mass spectrometry using hollow nanomechanical resonators. Scientific Reports, 7, 3535.
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