Filter retardation assay for protein detection

Filter retardation assay (FRA) is based on filtration of proteins on a cellulose acetate membrane with pores of 0.22 µm by a vacuum applied at a Bio-Dot SF Microfiltration Apparatus (Bio-Rad, Hercules, CA, USA). FRA allows the detection of insoluble protein species larger than the pores of the membrane. The following amounts of proteins were loaded: 1.5 µg of NSC34 PBS and NP-40 soluble extracts (NP-40 insoluble extracts were loaded as equal volume), 6 µg of C2C12 PBS and NP-40 soluble extracts (NP-40 insoluble extracts were loaded as equal volume). After vacuum filtration proteins were fixed on the membrane with 20% methanol and then the membrane was incubated for 1 hr at RT in blocking solution (5% of dried non-fat milk (Euroclone) in TBS-T 1X) and then with primary antibody for 1 hr at RT. After two washes with TBS-T 1X the membrane was incubated with the secondary antibody (find incubation timing and antibody dilution below). After three washes with TBS-T 1X at RT membrane was incubated with ClarityTM Western ECL Blotting Substrate (Bio-Rad) to reveal the signal. Images were acquired using a ChemiDoc XRS System (Bio-Rad)^{36 (link)}.

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Cicardi M.E., Cristofani R., Rusmini P., Meroni M., Ferrari V., Vezzoli G., Tedesco B., Piccolella M., Messi E., Galbiati M., Boncoraglio A., Carra S., Crippa V, & Poletti A. (2018). Tdp-25 Routing to Autophagy and Proteasome Ameliorates its Aggregation in Amyotrophic Lateral Sclerosis Target Cells. Scientific Reports, 8, 12390.

Publication 2018

Bio-Dot SF Microfiltration Apparatus ClarityTM Western ECL Blotting Substrate ChemiDoc XRS System

Antibody Assay Blotting western Cellulose acetate Dilution Filtration Membrane Methanol Milk Np 40 Proteins Vacuum

Corresponding Organization : University of Florence

Other organizations : University of Milan, University of Modena and Reggio Emilia

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Variable analysis

independent variables

Amount of protein loaded: 1.5 µg of NSC34 PBS and NP-40 soluble extracts, 6 µg of C2C12 PBS and NP-40 soluble extracts

dependent variables

Detection of insoluble protein species larger than the pores of the 0.22 µm cellulose acetate membrane

control variables

Pore size of the cellulose acetate membrane (0.22 µm)
Vacuum applied at a Bio-Dot SF Microfiltration Apparatus (Bio-Rad, Hercules, CA, USA)
Incubation time of the membrane in blocking solution (1 hr at RT)
Incubation time of the membrane with primary antibody (1 hr at RT)
Incubation time of the membrane with secondary antibody (not explicitly mentioned)
Dilution of secondary antibody (not explicitly mentioned)
Washing steps with TBS-T 1X (two washes after primary antibody, three washes after secondary antibody)

controls

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

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