To monitor protein levels, frozen muscles were pulverized by means of Qiagen TissueLyser and protein extracts were prepared in an appropriate buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 10% glycerol, 2% SDS, 1% Triton X-100, Complete EDTA-free protease inhibitor mixture (Roche), 1 mM PMSF, 1 mM NaVO3, 5 mM NaF, and 3 mM β-glycerophosphate. 40 μg of total proteins was loaded, according to BCA quantification. Proteins were separated by SDS-PAGE electrophoresis, in commercial 4-12% acrylamide gels (Thermo Fisher Scientific), and transferred onto nitrocellulose membranes (Thermo Fisher Scientific) by semidry electrophoretic transfer. Blots were blocked for 1 hour at RT with 5% nonfat dry milk (Bio-Rad) in TBS-tween (0.5 M Tris, 1.5 M NaCl, and 0.01% Tween) solution and incubated at 4°C with primary antibodies. Secondary antibodies were incubated 1 hr at RT. The following primary antibodies were used: anti-LC3 (1 : 1000, Cell Signaling), anti-p62 (1 : 5000, Sigma-Aldrich), anti-actin (1 : 20000, Santa Cruz), and anti-MCU (1 : 1000 Sigma-Aldrich). Secondary HRP-conjugated antibodies were purchased from Bio-Rad and used at 1 : 5000 dilution.
Acrylamide gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 4–12% acrylamide gel is a laboratory equipment product used for protein separation and analysis. It is a polyacrylamide gel with a gradient concentration of 4% to 12% acrylamide. The gel is designed to provide effective separation of a wide range of protein sizes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (3 mentions)
Non fat dry milk, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Anti mcu, by Merck Group (2 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Iblot system, by Thermo Fisher Scientific (2 mentions)
Phosstop, by Roche (2 mentions)
Western lightning ecl, by PerkinElmer (2 mentions)
β actin, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Tissuelyser, by Qiagen (1 mentions)
Anti lc3, by Cell Signaling Technology (1 mentions)
Anti p62, by Merck Group (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Anti tom20, by Santa Cruz Biotechnology (1 mentions)
Anti micu2, by Merck Group (1 mentions)
Anti grp75, by Santa Cruz Biotechnology (1 mentions)
Irdye 680 or 800, by LI COR (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg, by LI COR (1 mentions)
19 protocols using acrylamide gel
1
Protein Extraction and Quantification
2
Mitochondrial Protein Quantification by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To monitor protein levels, protein extracts were prepared in RIPA buffer (125 mM NaCl, 25 mM Tris-Cl pH7.4, 1 mM EGTA-Tris pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and Complete EDTA-free protease inhibitor mixture-Roche-). 40 μg of total proteins were loaded, according to BCA quantification. Proteins were separated by SDS-PAGE electrophoresis, in commercial 4%–12% acrylamide gels (Thermo Fisher Scientific) and transferred onto nitrocellulose membranes (Thermo Fisher Scientific) by wet electrophoretic transfer. Blots were blocked 1 h at RT with 5% non-fat dry milk (Bio-Rad) in TBS-tween (0.5M Tris, 1.5M NaCl, 0.01% Tween) solution and incubated at 4°C with primary antibodies. Secondary antibodies were incubated 1 h at RT. The following antibodies were used: anti-MCU (1:1000, Sigma-Aldrich), anti-MICU1 (1:1000 Novus Biologicals), anti-MICU2 (1:1000 Sigma-Aldrich), anti-GRP75 (1:5000, Santa Cruz Biotechnology) and anti-TOM20 (1:20000, Santa Cruz Biotechnology). Secondary HRP-conjugated antibodies were purchased from Bio-Rad and used at 1:5000 dilution.
3
Western Blot Analysis of ZIKV Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer (Cell Signaling Technologies) with 1X protease inhibitor. 10 µg of protein samples were resolved under denaturing conditions on 4–12% acrylamide gels (Thermo Fisher Scientific). ZIKV NS4B, and NS2B, BRD4, TLN1, PNPLA6, Actin and GAPDH were detected using primary antibodies mentioned above. Goat anti-mouse IgG and goat anti-rabbit IgG coupled with IRDye 800 or 680 (LI-COR Biosciences) were used as secondary antibodies. Blots were developed in the LI-COR Odissey luminescence system and protein expression was quantified by densitometry with the Image Studio Lite Ver 5.0 software.
4
Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue and cells protein was extracted by homogenization in lysis buffer (Sigma). Twenty micrograms of total protein were resolved by SDS-PAGE on 4–12% acrylamide gels (Invitrogen) and transferred to PVDF membranes (Invitrogen), followed by immunoblotting with antibody. Quantification of bands on western blot was accomplished by scanning the blots, then determining the densities of the bands using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).
5
Detecting Misfolded Prion Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The PMSA-derived products were digested with proteinase K (Roche) and subjected to electrophoresis and staining of total protein to assess the presence of proteinase K-resistant misfolded PrP (rec-PrPres) in each sample. Briefly, 400 μl of each PMSA product were transferred to 1.5 ml Eppendorf tubes and proteinase K added to a final concentration of 25 μg/ml. The tubes were incubated at 42°C for 1 h and then centrifuged at 19,000 g at 4°C for 15 min. The supernatant was discarded carefully without disturbing the pellet and the pellet was washed with 500 μl of PBS and centrifuged again at 19,000 g for 5 min at 4°C. After removing all the PBS, the pellet was resuspended in 15 μl of loading buffer NuPage 4X (Invitrogen) diluted in PBS; at this point samples could be stored at −20°C until required. For electrophoresis, digested samples, together with non-digested controls, were boiled at 100°C for 10 min and loaded in 4–12% acrylamide gels (Invitrogen) for 1 h 20 min at 70 V for 10 min at 110 V for the next 10 min and 150 V for 1 h. The gel was then transferred to a glass bucket and total protein stained using BlueSafe (NzyTech) for at least 1 h at room temperature with gentle rocking.
6
Immunoprecipitation and Western Blot Analysis of MFG-E8
7
SDS-PAGE and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells (2 x 106) were washed with phosphate-buffered saline (PBS) and lysed in 100μl of SDS sample buffer (LifeTechnologies). Whole-cell extracts (15μl [from 3 x 105 cells]) were separated on 10% acrylamide gels (LifeTechnologies) or Phos-Tag gel (Wako Chemicals). Following electrophoresis, the gel was transferred to a PVDF membrane using an iBlot Gel Transfer system (LifeTechnologies)and stained using anti-SAMHD1 (Proteintech), anti-tubulin (Sigma-Aldrich), and anti-GAPDH (Santa Cruz Biotechnology) polyclonal antibodies.
8
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Non-specific cell lysis was performed in isolated human chondrocytes (passage 1, 2, and 5) by rinsing cells twice with ice cold PBS and then incubating them for 30 min on ice in RIPA buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 5 mM EDTA. The supernatant was extracted after centrifugation for 30 min at 13 000g and 4 °C, and mixed with 1 : 4 v/v of Laemmlli's buffer (Boston Bioproducts, Boston, MA) with 8% mercaptoethanol (Sigma). 10% acrylamide gels (Life Technologies) were used for protein separation. Gels were blotted using Life Technologies gel transfer stacks and blotting system. Membranes were blocked with 5% powdered milk and incubated overnight at 4 °C while shaking with the antibodies of interest at a 1 : 1000 dilution (Collagen I, II and Elastin, Abcam, Cambridge, USA). After two washes with PBS-T (PBS, 0.05% Tween 20, Sigma), membranes were incubated with appropriate HRP-conjugated secondary antibodies diluted 1 : 2000 for 1 h while shaking at room temperature. After two 10 min washes in PBS-T, LuminataTM Forte Western HRP Substrate (Milipore, Billerica, MA) was applied and luminescence was detected in a Chemidoc XRS + (Bio-Rad, Hercules, CA).
9
Western Blot Analysis of RcsF and Lpp
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples were separated in 12% or 4%–12% acrylamide gels (Life Technologies) and transferred to nitrocellulose or polyvinylidene fluoride membranes (Whatman, 0.45 μm). Immunoblotting was performed as described previously [11 (link)]. Rabbit anti-RcsF (from the Collet laboratory’s collection) and rabbit anti-Lpp (from the Hughes laboratory’s collection) were diluted 1:10,000 in 1% skim milk, TBS-T (50 mM Tris-HCl [pH 7.6], 0.15 M NaCl, 0.1% Tween 20). The membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma; 1:10,000 in 1% skim milk, TBS-T) and washed with dilution buffer. Labelled proteins were detected via enhanced chemiluminescence (Pierce ECL Western Blotting Substrate, Thermo Scientific), which was imaged with a GE ImageQuant LAS4000 camera (GE Healthcare Life Sciences). To measure RcsF levels, band intensities were quantified using ImageJ 1.48v (NIH) and analysed with ImageQuant TL software 1Dv8.1 to ensure that they were within the linear range.
10
Quantifying PTEN and AKT Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were lysed in 50 mM Tris–HCl, pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1% glycerol, 100 μM vanadate, protease inhibitor cocktail and PhosSTOP (Roche Diagnostics GmbH). Lysates were subjected to SDS-PAGE using 4–12% acrylamide gels (Life Technologies) and transferred to PVDF membranes using the iBlot system (Life Technologies). The proteins were detected by western blot analysis by using an enhanced chemiluminescence system (Western Lightning–ECL, PerkinElmer). Antibodies used were specific for PTEN (Cell Signaling, cat. no. 9552, 1:1000), AKT (Cell Signaling, cat. no. 9272, 1:1000), phospho-AKT (Cell signaling, cat. no. 4060S, 1:1000) and β-actin (Sigma-Aldrich, cat. no. A5441, 1:5000). Membranes were first incubated for p-AKT followed by reprobing the same membranes for total AKT and PTEN.
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!