ProtoGlow ECL


ProtoGlow ECL fits into any existing Western blot workflow using the same basic protocol as standard
ECL kits. Note: The extreme sensitivity of this substrate may require additional dilution of antibody
stocks and/or alterations in blocking protocols to avoid excessive background.
1. Prepare your protein blot. PVDF or nitrocellulose may be used.
2. Block membrane for 60 minutes at room temperature. Use BLOTTO, BSA or nonprotein blocking
3. Incubate blot with primary antibody for 60 minutes at RT with gentle agitation.
4. Wash blot in PBST or TBST as follows:
    a) One brief wash
    b) Once at 15 minutes with 1.0 mL/cm2 membrane
    c) Three times for 15 minutes with 0.5 mL/cm2 membrane per wash
5. Incubate blot with secondary antibody for 60 minutes at RT on an oscillating plate or rocker.
6. Wash blot three times in TBST for 5 minutes each with at least 0.5 mL/cm2 membrane per wash.
7. Make the ProtoGlow ECL reagent by mixing equal amounts of parts A and B. Provide 0.1-0.2 mL/cm2
membrane and place on blot for 2 minutes.
8. Drain excess reagent.
9. Cover damp blot with plastic wrap and place in cassette.
10. Insert film into cassette and expose. In most cases, 10-60 seconds is a sufficient exposure.
Catalog Number: 
Full Protocol Sheet: 

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