Whole retina extracts were prepared from eyes of age-matched PhLP1F/FCre+ mice and controls under ambient illumination. These retinas were harvested and placed in ice-cold RIPA buffer (phosphate buffered saline with 1% NP-40 and 6 μl/mL Sigma Protease inhibitor cocktail). The retinas were then passed through an 18G needle 20 times and a 25G needle 10 times to release the proteins. Extracts were centrifuged at 13,800 rpm for 10 min at 4°C to remove cellular debris. Protein concentrations were determined by BCA protein assay, and extracts with equal amounts of protein were resolved on 10% or 14% Tris-glycine-SDS gels or 16.5% Tricine-SDS gels and transferred onto nitrocellulose membranes using an iBlot transfer apparatus (Invitrogen). After blocking with LICOR Blocking buffer for 1 hr, membranes were immunoblotted for each visual protein of interest. The amounts of each protein in the immunoblots were quantified using a LICOR Odyssey near-infrared imaging system and compared to controls.
Iblot transfer apparatus
Manufactured by Thermo Fisher Scientific
Sourced in United States
The iBlot transfer apparatus is a rapid, semi-dry gel-to-membrane transfer system designed for efficient protein transfer from polyacrylamide gels to membranes. The system utilizes optimized transfer stacks and an electric current to facilitate the transfer process.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Sigma protease inhibitor cocktail, by Merck Group (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Western lightning plus ecl reagent, by PerkinElmer (1 mentions)
Nupage sample reducing agent, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
G box chemi xr5 system, by Syngene (1 mentions)
G box chemi system, by Syngene (1 mentions)
Phospho fak, by Cell Signaling Technology (1 mentions)
Phospho mek, by Cell Signaling Technology (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
E cadherin n cadherin, by Cell Signaling Technology (1 mentions)
C raf, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
Phospho p90rsk, by Cell Signaling Technology (1 mentions)
Ecl plus enhanced chemiluminescence reagents, by Cytiva (1 mentions)
Hsp90, by Cell Signaling Technology (1 mentions)
Itgb3, by Cell Signaling Technology (1 mentions)
10 protocols using iblot transfer apparatus
1
Retinal Protein Extraction and Quantification
2
Protein Extraction and Western Blot Analysis
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Cells were grown to 80% confluency and lysed with cell lysis buffer (Cell Signaling #9803) on ice for 30 min. Lysate was cleared by centrifuging at 10,000 rpm for 10 min and collecting the supernatant. 30 μg of protein lysate was separated on 4–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred to nitrocellulose membranes using the iBlot transfer apparatus (Invitrogen). Primary antibodies used included glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology Inc., 25778), CHD1 (Novus Biologicals, NB100-60411), focal adhesion kinase (FAK) (Cell Signaling Technologies or CST, 3285P), phosphorylated extracellular signal regulated kinase (pErk) (CST, 4370S), total Erk (CST, 9102S), phosphorylated protein kinase B (pAKT) (CST, 4060S), total AKT (CST, 72), SPARC (CST, 8725), pMEK 1/2 (mitogen-extracellular signal-regulated kinase 1/2) (CST, 9154), total MEK 1/2 (CST, 9126), tenascin C (CST, 12221), and vitronectin (CST, 60896). Secondary antibodies included horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Calbiochem, 401315). Clarity™ enhanced chemiluminescent (ECL) reagent (Bio-Rad, 102030712) was used as a substrate for western blots. Images were obtained with a Bio-Rad imager, and signals were quantitated using Bio-Rad Image Lab software.
3
Protein Separation and Immunoblotting
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Samples for SDS-PAGE analysis were prepared using NuPAGE LDS Sample Buffer (4x dilution) and NuPAGE Sample Reducing Agent (10x dilution) according to the manufacturer's instructions (Invitrogen). Protein samples were denatured by incubation at 70 °C for 10 min. Proteins were loaded at 0.5 μg per well for 12-well gels, and 0.375 μg per well for 15-well gels. HiMark™ pre-stained protein standards (31–460 kDa) were used as molecular mass markers. Electrophoresis was carried out on 1 mm NuPAGE NOVEX 3–8% Tris-Acetate Gels using NuPAGE Tris-Acetate SDS Running Buffer at 150 V for 70 min. Following electrophoretic separation, proteins were analysed using immunoblotting.
For immunoblotting, proteins were electroblotted onto PVDF membranes over a period of 7 min using an iBlot® transfer apparatus (Invitrogen). The membranes were blocked with 1% (w/v) BSA in TBS with Tween 20 (TBST) for 1 h and then incubated with primary antibody (A17, 1:10,000 and anti-dityrosine, 1:500) in blocking solution overnight at 4 °C. Membranes were then rinsed 3 times with TBST for 5 min before incubation with secondary HRP-conjugated antibody. Unbound antibody was removed by 4 washes with TBST for 10 min, and once with TBS for 10 min. Immune complexes were detected using Western Lightning Plus ECL reagent (NEL104001EA; Perkin Elmer, Waltham, MA, USA) and imaged using a G: BOX Chemi XR5 system (Syngene).
For immunoblotting, proteins were electroblotted onto PVDF membranes over a period of 7 min using an iBlot® transfer apparatus (Invitrogen). The membranes were blocked with 1% (w/v) BSA in TBS with Tween 20 (TBST) for 1 h and then incubated with primary antibody (A17, 1:10,000 and anti-dityrosine, 1:500) in blocking solution overnight at 4 °C. Membranes were then rinsed 3 times with TBST for 5 min before incubation with secondary HRP-conjugated antibody. Unbound antibody was removed by 4 washes with TBST for 10 min, and once with TBS for 10 min. Immune complexes were detected using Western Lightning Plus ECL reagent (NEL104001EA; Perkin Elmer, Waltham, MA, USA) and imaged using a G: BOX Chemi XR5 system (Syngene).
4
Western Blot Protein Analysis
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For western blotting, cell lysates were first prepared using RIPA buffer (Sigma-Aldrich, St. Louis, MO) in accordance with the manufacturer’s instructions. Protein samples were then applied to the wells of NuPAGE 4%–20% Tris-Gly gel, electrophoresed in sodium dodecyl sulfate running buffer (Invitrogen), and transferred to nitrocellulose membranes using the iBlot transfer apparatus (Invitrogen). Membranes were blocked in Tris-buffered saline containing 0.5% Tween 20 (TBS-T) and 5% bovine serum albumin (BSA) for 1 hour at room temperature, followed by incubation with the primary antibody overnight at 4°C. On the following day, after the membranes were washed three times for 10 minutes each in TBS-T, horseradish peroxidase–conjugated secondary antibody (Bio-Rad, Hercules, CA) in TBS-T containing 2% BSA was applied for 1 hour at room temperature. Proteins were visualized with ECL Plus enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ) using G-box Chemi Systems (SynGene, Bengaluru, India). The commercial antibodies used in this study were ERK, phospho-ERK, MEK, phospho-MEK, AKT, phospho-AKT, c-Raf, tetraspanin 7 (TSPAN7), E-cadherin N-cadherin, cleaved poly(ADP-ribose) polymerase (PARP), PARP, ITGB3, ITGAv, HSP90, Phospho-p90RSK, RSK, phospho-FAK and β-actin (all purchased from Cell Signaling Technology).
5
Immunoblot Analysis of NFAM1 Protein
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Cells were lysed in RIPA buffer (Thermo Fisher Scientific) with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific) and Phosphatase Inhibitor Cocktail (Sigma). Protein content was determined via Bradford protein assay kit (Thermo Fisher Scientific). Samples were run on NuPAGE, 4-12%, Bis-Tris, Mini Protein Gel and then transferred to an iBlot transfer stack using an iBlot transfer apparatus (Invitrogen) according to the Invitrogen Western Blot Technical Guide. Detection was performed according to the Li-COR Odyssey Western Blot Protocol, using α-human NFAM1 primary antibody (Sigma, cat#HPA031812) and donkey α-rabbit secondary antibody in IRDYE 800CW (LI-COR) in combination with an Odyssey Infrared Imager (LI-COR).
6
Protein Extraction and Immunoblotting Protocol
7
Western Blot Protein Detection Protocol
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Cellular extracts were quantified by Bradford (Sigma), and 15 μg were used for the assay. Proteins were transferred onto a PVDF membrane by wet-transfer performed at 30 V for 1 hour, or by dry-transfer using the iBlot Transfer Apparatus (Invitrogen). The membrane was blocked with 5% non-fat dry milk in 0.01% Tween-20 TBS (TBS-T) for 1 hour, incubated with primary antibody overnight at 4°C, and washed three times with TBS-T for 5 min. The membrane was incubated with the peroxidase-conjugated secondary antibody in TBS-T containing 1% non-fat dry milk for 1 hour at room temperature, and washed three times with TBS-T for 5 min. Proteins were detected using the Pierce ECL Western Blotting Substrate (Thermo Fisher) on a Bio-Rad ChemiDoc MP Imager, or using a chemiluminescence Amersham Hyperfilm MP (Cytiva). Primary and secondary antibodies were diluted in TBS-T and 1% non-fat milk as follows: anti-GFP (1 : 5000), anti-Ag85 (1 : 5000), anti-6X-His (1 : 2000), HRP-conjugated anti-His (1 : 2000); HRP-conjugated anti-rabbit (1 : 10000) and HRP-conjugated anti-mouse (1 : 10000).
8
Quantification of Chlamydia Protein Levels
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siRNA transfected cells were infected with Chlamydia as described above. Samples were separated on an SDS-12%PAGE and transferred using an iBlot transfer apparatus (Life Technologies). Primary antibodies used in this study include: rabbit anti-syntaxin 10 (Abgent; SanDiego, CA), mouse anti-GAPDH (EMD Millipore; Darmstadt, Germany), rabbit anti-Hc1 (Ted Hackstadt), rabbit anti-OmcB (Thomas Hatch, University of Tennessee Health Science Center, Memphis, TN), and mouse anti-HSP60 (Rick Morrison, Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR). The primary antibodies were detected using appropriate anti-mouse or anti-rabbit IgG secondary antibodies conjugated with IRDye 700 or IRDye 800 dyes (LI-COR). The blots were scanned and analyzed by densitometry with an Odyssey Infrared Imaging System using Image Studio imaging software (LI-COR). To quantitate OmcB and Hc1 protein levels, densitometry values were normalized to cHSP60, which was first normalized to host GAPDH. Data are representative of two independent experiments and results are expressed as mean and standard error of the mean, calculated by GraphPad Prism 6 software.
9
CCHFV and PRRSV vOTU Cleavage Assays
10
Western Blot Analysis of Membrane Proteins
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