Cells were harvested at 800 ×g for 10 min at 4 °C and washed once with ice-cold phosphate-buffered saline (PBS). Samples were then re-suspended in 20 μl 2 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer, heated to 95 °C for 10 min, and then subjected to SDS-PAGE. Separated proteins were then electroblotted onto Immobilon-P membrane (Millipore Corp.). Membranes were then blocked with 5% skimmed milk in TBS (137 mM NaCI, 2.7 mM KCI, 25 mM Tris base, pH 7.4, 0.2% Tween 20) for 1 h at room temperature. Probing with the primary antibody (mouse anti-HA epitope immunoglobulin G (IgG) at 1:10,000 dilution; Santa Cruz Biotechnology) was then carried out overnight at 4 °C. Membranes were washed twice with TBS and probed with secondary antibody (rabbit anti-mouse peroxidase-conjugate at 1:10,000 dilution) for 1 h at room temperature. Bound antibodies were detected by enhanced chemiluminescence using Biomax MR-1 films (Kodak). Films were scanned and, where relevant, quantitated using ImageJ software (NIH).
Biomax mr 1 film
Manufactured by Kodak
Sourced in United States
The BioMax MR-1 film is a laboratory product designed for autoradiography and other scientific imaging applications. It is a high-performance, double-emulsion film that provides high resolution and sensitivity for analyzing radioactive samples. The film is suitable for a variety of scientific research and analytical techniques, but its core function is to capture and record images of radioactive materials.
Automatically generated - may contain errors
8 protocols using biomax mr 1 film
1
Western Blot Analysis of HA-tagged Proteins
2
Niclosamide Modulates Wnt Pathway Proteins
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The effect of Niclosamide on protein expression of various downstream proteins of the Wnt signaling pathway was studied by using Western blotting technique [21] . After treatment with different concentrations of Niclosamide for 24 hrs in HGC-27 cell line, cells were collected and washed twice with 1X PBS, then lysed in Radioimmunoprecipitation assay (RIPA) buffer with Protease cocktail mixture & phosphatase inhibitor under gentle rotation for 20 min at 40C, and the supernatant was collected by centrifugation at 12,500g for 20 min at 40C and stored at -200C or used immediately. Protein concentration was determined by the BioRad Protein detection reagent (BioRad). Equal amounts of protein were separated on SDS polyacrylamide gels. The separated proteins were blotted or transferred onto polyvinyl difluoride (PVDF) membrane (PolyScreen, NEN Life Sciences, USA) and following blocking (5% nonfat dry milk in TBS-Tween (0.1%), and probed with relevant primary and HRP-conjugated secondary antibody. Membranes were visualized on Kodak BioMax MR-1 films, and further, the protein bands were subjected to a densitometry analysis using ImageJ software. The membranes were reprobed with β-actin antibodies as an internal control and to ensure equal loading. Each Western blot shown is representative of at least three independent experiments.
3
Ascorbate Detection in Biological Samples
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The extracts were centrifuged at 2000g for 5 min, and samples (50 µl) of the supernatant were run by HVPE (high-voltage paper electrophoresis) in pH 6.5 buffer (pyridine/ acetic acid/ H2O, 33:1:300 v/v/v) for 30 min at 2.5 kV or in pH 2.0 buffer (formic acid/acetic acid/ H2O, 1:4:45, v/v/v) for 50 min at 2.5 kV.
The presence of [14C]ascorbate and any degradation products formed was detected by autoradiography (3 week exposure to Kodak BioMax MR-1 film) or scintillation counting. Samples dried onto paper were assayed in 2 ml ScintiSafe scintillation fluid in a Beckman LS 6500 CE multi-purpose scintillation counter.
The presence of [14C]ascorbate and any degradation products formed was detected by autoradiography (3 week exposure to Kodak BioMax MR-1 film) or scintillation counting. Samples dried onto paper were assayed in 2 ml ScintiSafe scintillation fluid in a Beckman LS 6500 CE multi-purpose scintillation counter.
4
Quantification of Radioactive Samples
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Aqueous and ethanolic solutions were assayed for 14C by scintillation counting in 10 volumes of OptiPhase HiSafe 3 (PerkinElmer, Inc.) aqueous-miscible scintillant.
Gel electrophoretograms and TLCs were dried, then exposed to Kodak BioMax MR-1 film in the dark for ~4 weeks. Dry strips cut from paper chromatograms and paper electrophoretograms, and spots (localized by autoradiography) excised from TLCs, were assayed for 14C by scintillation counting in 2 mL of an aqueous-immiscible scintillant (Gold Star; Meridian Biotechnologies Ltd). Radioactive spots excised from gel electrophoretograms were hydrolysed in 2m trifluoroacetic acid at 100 °C for 1 h, then assayed for 14C by scintillation counting in 10 volumes of aqueous-miscible scintillant.
Gel electrophoretograms and TLCs were dried, then exposed to Kodak BioMax MR-1 film in the dark for ~4 weeks. Dry strips cut from paper chromatograms and paper electrophoretograms, and spots (localized by autoradiography) excised from TLCs, were assayed for 14C by scintillation counting in 2 mL of an aqueous-immiscible scintillant (Gold Star; Meridian Biotechnologies Ltd). Radioactive spots excised from gel electrophoretograms were hydrolysed in 2
5
Electrophoretic Separation of Ascorbate Compounds
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Aqueous samples were loaded onto Whatman No. 3 paper and electrophoresed at 2.5–3.5 kV for 30–70 min in a buffer of pH 2 (formic acid/acetic acid/water, 1 : 4 : 45, v/v/v) or pH 6.5 (pyridine/acetic acid/water, 33 : 1 : 300, v/v/v) [75 ].
Orange G (2 µl, 10 mM) was added to all samples as an internal marker, and electrophoretic mobilities (mOG) are calculated relative to orange G. Neutral compounds move a small distance away from the origin owing to electro-endo-osmosis, so mobilities were calculated with a neutral marker (e.g. DHA) as mOG = 0. After long electrophoresis runs, orange G ran off the paper, and mThrR [mobility relative to threarate (mThrR = 1.0) and glucose (mThrR = 0.0)] was used instead of mOG. Ascorbate-related compounds were stained with AgNO3 [76 ]. Paper electrophoretograms containing 14C-labelled compounds were exposed to photography film (Kodak BioMax MR-1 film) for 7 days.
Orange G (2 µl, 10 mM) was added to all samples as an internal marker, and electrophoretic mobilities (mOG) are calculated relative to orange G. Neutral compounds move a small distance away from the origin owing to electro-endo-osmosis, so mobilities were calculated with a neutral marker (e.g. DHA) as mOG = 0. After long electrophoresis runs, orange G ran off the paper, and mThrR [mobility relative to threarate (mThrR = 1.0) and glucose (mThrR = 0.0)] was used instead of mOG. Ascorbate-related compounds were stained with AgNO3 [76 ]. Paper electrophoretograms containing 14C-labelled compounds were exposed to photography film (Kodak BioMax MR-1 film) for 7 days.
6
In Situ Hybridization for Neurotransmitter Receptors
7
Quantifying Muscarinic Receptor Levels
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The protein levels of M2 and M3 were examined using western blot and five bladder specimens from each group were examined. The frozen tissue was pulverized and homogenized at 4°C. Homogenates were centrifuged at 13 000 rpm for 20 minutes at 4°C, the pellet discarded, and the supernatant was used immediately. After electrophoresis, the proteins were transferred to polyvinylidene fluoride membranes for 2 hours. Nonspecific binding sites were blocked by incubation for 1 hour 1 Tween 20/Tris-buffered saline containing 5% skim milk and 0.1% Tween 20. Membranes were then incubated with primary antibodies overnight. The primary antibodies anti-mAChR2 and anti-mAChR3 purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA, 1:100). Membranes were incubated with rabbit immunoglobulin G secondary antibody horseradish peroxidase (1:2000; Abcam Plc) for 1 hour after washing. Membranes were also probed with an anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody. Then, protein signals were detected by exposure on BioMax MR-1 film (Kodak, Rochester, NY, USA). Expression levels were calculated by measuring the density of the bands. The protein bands were quantified as a ratio to GAPDH using Image-Pro Plus.
8
Two-Dimensional Paper Electrophoresis of Labeled Compounds
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Electrophoresis was performed on Whatman No. 3 paper at 2.5-3.5 kV for 30-70 minutes in a buffer of pH 2.0 (formic acid/acetic acid/water, 1:4:45, v/v/v) or pH 6.5 (pyridine/acetic acid/ water, 33:1:300 v/v/v) (Fry, 2011) . Orange G (2 µl, 10 mM) was added to all samples as an internal marker. Neutral compounds move a small distance away from the origin owing to electro-endo-osmosis. DKG and related compounds were stained with AgNO3 (Fry, 2000) .
Electrophoretograms containing 14 C-labelled compounds autoradiographed on film (Kodak BioMax MR-1 film) for 7 days.
For 2-dimensional paper electrophoresis, a single sample, along with external markers, was loaded onto Whatman No. 3 paper and subjected to electrophoresis as described above. The paper was then dried before the lane of interest was cut out. This lane was then sewn onto the origin of a new sheet of Whatman No. 3 paper, so that the compounds in the sample lane were lined up along the new origin. Further markers (internal marker Orange G, and external marker DKG products) were added. This new paper was then subjected to electrophoresis at either pH 2.0 or pH 6.5 as described previously.
Electrophoretograms containing 14 C-labelled compounds autoradiographed on film (Kodak BioMax MR-1 film) for 7 days.
For 2-dimensional paper electrophoresis, a single sample, along with external markers, was loaded onto Whatman No. 3 paper and subjected to electrophoresis as described above. The paper was then dried before the lane of interest was cut out. This lane was then sewn onto the origin of a new sheet of Whatman No. 3 paper, so that the compounds in the sample lane were lined up along the new origin. Further markers (internal marker Orange G, and external marker DKG products) were added. This new paper was then subjected to electrophoresis at either pH 2.0 or pH 6.5 as described previously.
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