Reactions were performed exactly as for invasion assays using 50 ng of pTel plasmid (0.6 nM final concentration), the indicated amounts of recombinant proteins and 10 nM 5’-(UUAGGG)5-3’ oligonucleotides. Reactions were stopped by adding 4 μl of 6X gel-loading buffer (30% glycerol, 0.3% bromophenol blue, 0.3% xylene cyanol). Products were fractionated by electrophoresis in 1% agarose gels followed by incubation in SDS PAGE running buffer (25 mM Tris-Cl, 192 mM glycine, 0.1% SDS) for 5 min at room temperature and transfer to nitrocellulose membranes (Maine Manufacturing LLC) using a Trans-Blot SD Semi-Dry Transfer Cell apparatus (Bio-Rad). Western blots were performed as above. Signals were acquired using a FluorChem HD2 apparatus (Alpha Innotech) and quantified using ImageJ. Statistical analysis was performed in Microsoft Excel. The number of independent experiments (n) is indicated in figure legends.
Trans blot sd semi dry transfer cell apparatus
Manufactured by Bio-Rad
Sourced in United States
The Trans-Blot SD Semi-Dry Transfer Cell apparatus is a laboratory equipment used for the transfer of proteins from polyacrylamide gels to membranes for further analysis. It provides a fast, efficient, and consistent method for the transfer process, which is a critical step in various biochemical and molecular biology techniques.
Automatically generated - may contain errors
Lab products found in correlation
Fluorchem hd2 apparatus, by Bio-Techne (4 mentions)
Gtx117624, by Abcam (2 mentions)
Gtx111677s, by GeneTex (2 mentions)
Hrp conjugated goat anti mouse and anti rabbit iggs, by Fortis Life Sciences (2 mentions)
Hybond lfp pvdf membrane, by GE Healthcare (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
6 protocols using trans blot sd semi dry transfer cell apparatus
1
Telomeric DNA Binding Assay
2
Promastigote Lysis and Western Blot
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Late log-phase promastigotes were washed with ice-cold PBS containing 1 mM Na3VO4, then lysed in 50 mM Tris-HCl pH 8, 150 mM NaCl and 1% Nonidet P-40, containing complete protease inhibitors (Roche Applied Science) and phosphatase inhibitors (1 mM Na3VO4, 50 mM NaF, 1.5 mM EGTA and 10 mM Na4P2O7). Samples were sonicated briefly, and insoluble material was removed by centrifugation for 10 min at 4°C. Protein concentrations were determined using the Pierce BCA protein assay kit (Pierce). Proteins were separated by SDS-PAGE and then transferred to Hybond-LFP PVDF membranes (GE Healthcare Life Sciences) using a Trans-Blot SD Semi-Dry Transfer Cell apparatus (BioRad). Membranes were blocked with 5% BSA and incubated with the mouse monoclonal antibody CA7AE (MediMabs). For immunodetection, goat anti-mouse IgM Heavy Chain Secondary antibody conjugated with horseradish peroxidase (HRP), and enhanced chemiluminescence (ECL) detection reagents from GE Healthcare Life Sciences were used.
3
Telomeric DNA Binding Assay
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Reactions were performed exactly as for invasion assays using 50 ng of pTel plasmid (0.6 nM final concentration), the indicated amounts of recombinant proteins and 10 nM 5’-(UUAGGG)5-3’ oligonucleotides. Reactions were stopped by adding 4 μl of 6X gel-loading buffer (30% glycerol, 0.3% bromophenol blue, 0.3% xylene cyanol). Products were fractionated by electrophoresis in 1% agarose gels followed by incubation in SDS PAGE running buffer (25 mM Tris-Cl, 192 mM glycine, 0.1% SDS) for 5 min at room temperature and transfer to nitrocellulose membranes (Maine Manufacturing LLC) using a Trans-Blot SD Semi-Dry Transfer Cell apparatus (Bio-Rad). Western blots were performed as above. Signals were acquired using a FluorChem HD2 apparatus (Alpha Innotech) and quantified using ImageJ. Statistical analysis was performed in Microsoft Excel. The number of independent experiments (n) is indicated in figure legends.
4
Western Blot Analysis of Telomeric Proteins
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Total proteins prepared in 2X Laemmli buffer were separated in 8-10% polyacrylamide gels and transferred to nitrocellulose membranes (Maine Manufacturing LLC) using a Trans-Blot SD Semi-Dry Transfer Cell apparatus (Bio-Rad). Western blots were performed according to standard procedures using the following primary antibodies: a mouse monoclonal anti-TRF2 (Millipore, 05-521, dilution 1:2000), a rabbit polyclonal anti-TRF1 raised against full-length recombinant TRF1 (kind gift from Jan Karlseder, 1:2000), a rabbit polyclonal anti-Tankyrase 1 (kind gift from Susan Smith, 1:5000), a rabbit polyclonal anti-RNaseH1 (GeneTex, GTX117624, 1:1000), a mouse monoclonal anti-hbetaActin (Abcam, ab8224, 1:10000), and a rabbit polyclonal anti-LaminA (GeneTex, GTX111677S, 1:5000). Secondary antibodies were HRP-conjugated goat anti-mouse and anti-rabbit IgGs (Bethyl Laboratories, 1:5000). Signals were acquired using a FluorChem HD2 apparatus (Alpha Innotech).
5
Western Blot Analysis of Telomeric Proteins
6
Western Blot Analysis of Influenza H5 Antigen
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H5 HA (A/Anhui/1/05) was applied to 12% SDS-PAGE and transferred to a PVDF membrane with a Trans-Blot SD semi-dry transfer cell apparatus (Bio-Rad, USA). hH5M9 (1 mg/ml) was used as primary antibody. A rabbit polyclonal antibody against A/Anhui/1/05 was used as the positive control. IRDye 800CW-conjugated goat anti-human or goat anti-rabbit IgG (LI-COR, USA) was used as the secondary antibody for visualization. The fluorescent bands were detected by Odyssey Imaging System (LI-COR, USA).
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