iBlot™ 3 Transfer Stacks, mini, low-fluorescence PVDF - FAQs

Yes, the low fluorescence PVDF membrane included with the iBlot 3 Transfer Stacks, low fluorescence, PVDF (Cat. No. IB34003 (midi) and Cat. No. IB34004 (mini)) performs similarly to standard PVDF in chemiluminescent applications.

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To minimize membrane autofluorescence, the PVDF membranes are packaged dry. Hence, an activation step must be performed prior to use. If autofluorescence is not a concern, we recommend using iBlot 3 Transfer Stacks, PVDF (Cat. No. IB34001 (midi) or Cat. No. IB34002 (mini)) which contain pre-activated PVDF membranes already incorporated into the transfer stack.

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To minimize membrane autofluorescence, the PVDF membranes are packaged dry. Hence, an activation step must be performed prior to use. If autofluorescence is not a concern, we recommend using iBlot 3 Transfer Stacks, PVDF (Cat. No. IB34001 (midi) or Cat. No. IB34002 (mini)) which contain pre-activated PVDF membranes already incorporated into the transfer stack.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

We recommend wetting PVDF in 100% methanol or ethanol for 3 min and then rinsing with deionized (DI) water before use. Wetting for shorter times can result in incomplete activation, leading to inconsistent protein binding. Wetting for a longer time will not have a negative impact.

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PVDF is a hydrophobic membrane that will not readily interact or wet in water. To allow protein transfer and binding, the membrane must be initially wetted in methanol or ethanol and then rinsed with deionized (DI) water to allow protein binding to occur during transfer.

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If the PVDF membrane dries out after transfer and before immunodetection, the membrane can be re-wetted by soaking the membrane in 100% methanol or ethanol for 3 min and then rinsing with deionized (DI) water before proceeding to the blocking step. This re-wetting will generally not negatively impact protein binding.

If the PVDF membrane dries out before imaging, for fluorescent western blots, it can typically be imaged dry without re-wetting. However, note that some fluorescent dyes are prone to degradation which can accelerate when the membrane is dry. If wetting is desired, soak the membrane in 100% methanol or ethanol for 3 min and then rinse with DI water and proceed to imaging.

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Yes. For best stripping and re-probing results, we recommend air drying the membrane after the transfer prior to immunoblotting (it will require rehydration in 100% methanol or ethanol prior to immunoblotting). Drying helps to fix the transferred proteins onto the membrane, helping ensure they remain immobilized and do not diffuse or wash away during subsequent steps. Drying the membrane can improve the binding efficiency of antibodies during the blocking and probing stages, resulting in better signal detection and stronger, clearer bands.

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We recommend storing Invitrogen Low Fluorescence PVDF Membranes in the original container at room temperature, kept dry and away from moisture.

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We recommend using Blocker FL Fluorescent Blocking Buffer (10X) (Cat. No. 37565) and Fluorescent Compatible Sample Buffer (Cat. No. LC2570) for fluorescent western blots. We also recommend loading 5- to 10-fold less pre-stained molecular weight markers to avoid oversaturating the blot with fluorescent signal from the protein ladder.

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Yes, Invitrogen Low Fluorescence PVDF Membranes perform very well for both fluorescent and chemiluminescent detection.

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The optimal protein load amount is dependent on the target protein and expression abundance. To be in the linear range in a western blot, we typically recommend cell lysate load amounts of 1-5 µg for higher-abundance targets, and 3-30 µg for low-abundance targets. Note: For quantitative analysis, it is important that western blot results are in a linear range without membrane saturation.

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We recommend handling Invitrogen Low Fluorescence PVDF Membranes with gloves. Activate the membrane in 100% methanol or ethanol for 3 min, followed by a rinse with deionized (DI) water or transfer buffer.

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Invitrogen Low Fluorescence PVDF Membranes have a narrow distribution 0.3 µm pore size which is effective for transfer of both small and large molecular weight proteins with the appropriate transfer conditions and gel types.

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Yes. Invitrogen Low Fluorescence PVDF Membranes exhibit low background when excited with wavelengths ranging from UV to IR, making them compatible with fluorescent detection from 280 to 800 nm.

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By reducing membrane background noise, signal-to-noise ratio is increased, allowing more accurate data and less data correction. The low autofluorescence of the membrane also allows for longer exposure times, which improves the limit of detection.

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Invitrogen Low Fluorescence PVDF membranes exhibit lower autofluorescence than nitrocellulose membranes for exceptional signal-to-noise and LOD, especially in lower wavelength channels such as 555 and 488 nm. The choice between PVDF and nitrocellulose will be protein dependent, as certain proteins may bind more strongly to one membrane type over the other.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.