Total RNA was prepared using TRIzol reagent (Invitrogen) from GIV-infected GK cells at a multiplicity of infection (m.o.i.) of 10. Ten micrograms of RNA were separated on a 1% formaldehyde agarose gel, and then transferred onto a Hybond-N membrane (Amersham Biosciences). The membrane was hybridized at 42°C overnight with a [32P]dCTP-radiolabeled GIV-CARD DNA probe, which was synthesized using GIV-CARD-F/GIV-CARD-R primer pairs (Table 1 ). After hybridization, the membrane was washed with a solution containing 0.1% SDS and 0.1× SSC, and subsequently exposed to Biomax X-ray film (Kodak) for signal detection. Control RNA was collected from mock-infected GK cells. Indicated cultures were pretreated for 1 h before infection with cycloheximide (CHX, final concentration of 200 μg/ml; Calbiochem) or aphidicolin (APH, final concentration of 5 μg/ml; Calbiochem), to inhibit protein or DNA synthesis, respectively.
Biomax x ray film
Manufactured by Kodak
Sourced in United States, Japan
Biomax X-ray film is a high-quality photographic film designed for use in various laboratory and scientific applications. It is a versatile product that captures detailed images with exceptional clarity and resolution. The film is suitable for a range of applications, including DNA sequencing, autoradiography, and general X-ray imaging procedures.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Hybond n membrane, by Cytiva (1 mentions)
Hrp conjugated anti rabbit secondary antibody, by Promega (1 mentions)
Pierce ecl western blotting substrate detection reagent, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Cytiva (1 mentions)
Ecl plus chemiluminescence kit, by Cytiva (1 mentions)
Phospho chk1 ser345, by Cell Signaling Technology (1 mentions)
Cyclin e, by Santa Cruz Biotechnology (1 mentions)
Phospho p53 ser15, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Ultrahyb oligo hybridization buffer, by Thermo Fisher Scientific (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Zeta probe membrane, by Bio-Rad (1 mentions)
5 protocols using biomax x ray film
1
RNA Northern Blot Analysis of GIV Infection
2
Quantitative Dot-Blot Assay for αAI
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Transformed colonies were transferred onto a replica plate and each colony was grown in 600 µL of YPCGal media in a 96-well microplate at 28 °C for 72–96 h at 250 rpm. Cleared supernatants (200 µL) were loaded into a dot blot apparatus connected to a vacuum pump. Pure Pinto-αAI [2 (link)] in sterile YPCGal (1 ng/µL) was used as positive control. Cleared YPCGal inoculated with untransformed cells (200 µL) and sterile YPCGal (200 µL) were used as negative controls. The nitrocellulose membrane was probed with a rabbit anti-αAI polyclonal antibody (1:10,000 dilution), and then probed with HRP-conjugated anti-rabbit secondary antibody (1:1000 dilution, Promega Corp. Madison, WI, USA). Protein antibody complexes were made visible using Pierce ECL Western Blotting Substrate detection reagent (Thermo-Fisher Scientific, Inc., Waltham, MA, USA) and autoradiography film (Kodak Biomax X-ray film). The relative amount of protein in dots was estimated by densitometry using ImageJ Software [38 ].
3
Western Blotting Protein Expression Analysis
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Western Blotting was used to analyze the levels of protein expression. Briefly, 15 µg of the protein from the whole-cell extract was separated by using 10% SDS-PAGE. The resolved proteins in the gel were transferred onto a polyvinylidene fluoride membrane (Amersham, GE Health, Sweden) at room temperature for 1.5 hr. The membrane was washed with 1× TBST and blocked with 2% bovine serum albumin (BSA) (for p21 and phospho-CHK1 analysis) or 10% nonfat milk (for other protein analysis) in 1× TBST for 1 hr. After washing, the membrane was incubated with primary antibodies in 1% BSA (for p21 and phospho-CHK1 analysis) or 5% nonfat milk (other protein analysis) in 1× TBST at 4°C overnight. The primary antibodies: Bmi-1 (F6; 1:600) was from Upstate, Millipore (Billerica, MA); p21WAF/Cip1 (1:200), cyclin E (1:1000) and actin (1:1000) were from Santa Cruz Biotechnology; p53 (1:1000), phospho-p53(ser15) (1:500), CHK1 (1:500) and phospho-CHK1(ser345) (1:500) were from Cell Signaling Technology (Danvers, MA). After washing twice with 1× TBST for 20 min, the membrane was incubated with appropriate secondary antibodies (1:3,000) in 5% nonfat milk in 1× TBST for 1 hr at room temperature. After washing, the membrane was incubated with ECL-Plus chemiluminescence kit (Amersham) for 5 min. The signals were visualized using Biomax X-ray film (Kodak, Rochester, NY).
4
Western Blot Analysis of Apoptosis Markers
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After treating cells as described above, total protein was extracted from cell lysates using RIPA buffer, and protein concentrations were determined with a BCA protein quantitative analysis kit. A total of 40 µg of protein was loaded onto a 15% SDS-polyacrylamide gel for electrophoresis (PAGE) and then transferred to a nitrocellulose membrane (Whatman, New Jersey, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline (pH 7.4) with 0.1% Tween-20 for 1 h at room temperature before overnight incubation with primary antibodies specific for α-Syn (1:1000), Bax (1:500), Bcl-2 (1:1000), and β-actin (1:1500) in 5% skim milk-Tris buffered saline Tween (TBST). The membranes were then rinsed 3 times in TBST and incubated with horseradish peroxidase-conjugated secondary IgG (1:2000) in TBST for 1.5 h, followed by a final series of rinses in TBST. The resulting bands of protein were visualized on Biomax X-ray film (Kodak, Japan) using the Super Signal West Pico Chemiluminescent Kit.
5
Northern Blot Analysis Protocol
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Northern blot analysis was performed as previously described (14) . Briefly, 10 μg of total RNA was loaded into each well of an 8% polyacrylamide-7M urea gel (USB Corporation) run at 300 V for 1.5 h in 1x TBE. The gel was transferred overnight to Zeta-Probe membrane (Bio-Rad) at 12 V at 4˚C. Following transfer, membranes were UV crosslinked and pre-hybridized in ULTRAhyb Oligo Hybridization Buffer (Invitrogen) for 1 h for at 45˚C. Oligonucleotides and is also made available for use under a CC0 license.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (which this version posted April 1, 2022. ; https://doi.org/10.1101/2022.04.01.486790 doi: bioRxiv preprint 8 complimentary to the target were end-labeled with γ-32 P-ATP by T4 polynucleotide kinase (New England Biolabs). Labeled oligonucleotides were added to the membrane and incubated overnight at 45˚C. Subsequently, membranes were rinsed 2X with 2× SSC + 0.1% SDS, 1X with 0.2× SSC + 0.1% SDS, washed with 0.2× SSC + 0.1% SDS at 45˚C for 25 min and then rinsed 1X with 0.2× SSC + 0.1% SDS. Membranes were exposed to KODAK Biomax X-ray film at -80˚C.
was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105
The copyright holder for this preprint (which this version posted April 1, 2022. ; https://doi.org/10.1101/2022.04.01.486790 doi: bioRxiv preprint 8 complimentary to the target were end-labeled with γ-32 P-ATP by T4 polynucleotide kinase (New England Biolabs). Labeled oligonucleotides were added to the membrane and incubated overnight at 45˚C. Subsequently, membranes were rinsed 2X with 2× SSC + 0.1% SDS, 1X with 0.2× SSC + 0.1% SDS, washed with 0.2× SSC + 0.1% SDS at 45˚C for 25 min and then rinsed 1X with 0.2× SSC + 0.1% SDS. Membranes were exposed to KODAK Biomax X-ray film at -80˚C.
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