Immunoblotting Analysis of Protein Interactions

Immunoblotting was carried out as previously described54 (link). Briefly, equal amounts of whole cell extracts (30 μg) or equal volumes of pull-down materials were analyzed by 10% SDS-PAGE under reducing conditions. The molecular weight of proteins was confirmed using prestained protein ladder (PageRuler, Fermentas). The R1(57) anti-APP C-terminal rabbit antibody55 (link), a kind gift of Dr. S. Efthimiopoulos (University of Athens, Greece) and the rabbit anti-AUF-1 antibody (07-260, Millipore) were used in a dilution 1:2000, the mouse anti-ELAVL and the rabbit anti-U2AF65 antibodies (3A2 and H300, respectively; all from Santa Cruz Biotechnology) in a dilution 1:1000 and the mouse anti-SAP97 (K64/15 UC Davis/NIH NeuroMab Facility and Antibodies Inc.) in a 1:50 dilution. Finally, the mouse anti-β-tubulin, the mouse anti-βΙΙΙ-tubulin (T5201 and T8660, respectively from Sigma) and the mouse anti-GAPDH-HRP conjugated (HRP-60004 from Proteintech) antibodies were used in a dilution 1:5000. All secondary HRP-conjugated antibodies (Santa Cruz Biotechnology) were used in a 1:5000 dilution. Each sample was tested in duplicate and samples obtained from three independent experiments were used for analysis. Densitometric analysis of immunoblotting images was performed using the image analysis software Image J (NIH, USA).

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Fragkouli A., Koukouraki P., Vlachos I.S., Paraskevopoulou M.D., Hatzigeorgiou A.G, & Doxakis E. (2017). Neuronal ELAVL proteins utilize AUF-1 as a co-partner to induce neuron-specific alternative splicing of APP. Scientific Reports, 7, 44507.

Publication 2017

PageRuler mouse anti-β-tubulin mouse anti-GAPDH-HRP conjugated (HRP-60004

Anti antibodies Anti antibody Antibodies Cell extracts Densitometric Dilution Equal Gapdh Immunoblotting analysis Mouse Protein Rabbit Sap97 Sds page Tubulin

Corresponding Organization :

Other organizations : Academy of Athens, Pasteur Hellenic Institute

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Immunoblotting Technique for Protein Analysis

Immunoblotting was carried out as previously described [26 (link)]. Briefly, equal amounts of whole-cell extracts or equal volumes of pull-down material were separated by 12% SDS-PAGE under denaturing conditions and transferred to nitrocellulose membrane (Protran; Amersham/Merck, St. Louis, MO, USA). The nitrocellulose membranes were probed with the appropriate primary antibodies after blocking with Tris-buffered saline (TBS) containing 5% nonfat milk and 0.1% Tween-20 for 1 h at RT. Except for the neutravidin-HRP conjugate diluted to a final concentration of 1:2000, all primary antibodies were diluted in blocking buffer at 1:1000. Secondary HRP-conjugated antibodies were used in a 1:2000 dilution. The immunoreactive bands were visualized with the enhanced chemiluminescence (ECL) method using the Clarity substrate (BioRad). Data obtained from at least three independent experiments are presented here.

Gourdomichali O., Zonke K., Kattan F.G., Makridakis M., Kontostathi G., Vlahou A, & Doxakis E. (2022). In Situ Peroxidase Labeling Followed by Mass-Spectrometry Reveals TIA1 Interactome. Biology, 11(2), 287.

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Variable analysis

independent variables

Equal amounts of whole cell extracts (30 μg)
Equal volumes of pull-down materials

dependent variables

Protein expression or abundance

control variables

Reducing conditions for SDS-PAGE
Prestained protein ladder (PageRuler, Fermentas) for molecular weight confirmation
Dilutions of primary and secondary antibodies

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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