Western Blot Analysis of Tat-Trx1 Protein

After transduction of Tat-Trx1 protein, protein extraction was performed using cell lysis buffer (RIPA; ELPIS BIOTECH, Daejeon, Korea) according to the manufacturer’s instructions. Then, equal amount of proteins were loaded into 15% SDS-PAGE and electrotransferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with TBS-T (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween 20, pH 7.5) buffer containing 5% non-fat dry milk or BSA for 1 h. After being washed with TBS-T buffer, the membrane was incubated with the indicated primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Then the membranes were washed with TBS-T buffer three times and the protein bands were identified using chemiluminescent reagents as recommended by the manufacturer (Amersham, Franklin Lakes, NJ, USA) (Shin et al., 2014 (link); Jegal et al., 2019 (link)).

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Yeo E.J., Eum W.S., Yeo H.J., Choi Y.J., Sohn E.J., Kwon H.J., Kim D.W., Kim D.S., Cho S.W., Park J., Han K.H., Lee K.W., Park J.K., Shin M.J, & Choi S.Y. (2021). Protective Role of Transduced Tat-Thioredoxin1 (Trx1) against Oxidative Stress-Induced Neuronal Cell Death via ASK1-MAPK Signal Pathway. Biomolecules & Therapeutics, 29(3), 321-330.

Publication 2021

RIPA

Antibodies Buffer Cell Horseradish peroxidase Membranes Milk Nacl Polyvinylidene difluoride Protein Ripa Sds page Tat protein Tris Trx1 Tween 20

Corresponding Organization :

Other organizations : Hallym University, Gangneung–Wonju National University, Soonchunhyang University, University of Ulsan, Ulsan College

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Variable analysis

independent variables

Transduction of Tat-Trx1 protein

dependent variables

Protein expression
Protein bands identified using chemiluminescent reagents

control variables

Equal amount of proteins loaded into 15% SDS-PAGE
Incubation with primary and secondary antibodies
Washing with TBS-T buffer

controls

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

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