Low Fluorescence PVDF Pre-cut Sheets, midi - FAQs

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16 product FAQs found

How long should I activate PVDF membrane in methanol?

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.

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

Why does PVDF need to be activated?

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.

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

My PVDF membrane has dried out. What should I do?

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.

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

What are the recommended washing steps to ensure low background and high signal-to-noise ratio when using Invitrogen Low Fluorescence PVDF Membranes?

The recommended wash condition after each step is: TBS or PBS with 0.05% Tween 20 for 5 min, repeated 3 times.

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

Can Invitrogen Low Fluorescence PVDF Membranes be stripped and re-probed, and if yes, how do I get the best results?

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.

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

What are the best practices or tips for transferring proteins onto Invitrogen Low Fluorescence PVDF Membranes to help ensure efficient and uniform binding?

Ensure uniform membrane pre-wetting in 100% methanol or ethanol. This activates the membrane and enhances protein binding. After activation, equilibrate the membrane in transfer buffer for at least 5 min to remove excess methanol and adapt it to the buffer conditions. Use clean labware and avoid excessive handling of the membrane to prevent contamination.

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

How should I store Invitrogen Low Fluorescence PVDF Membranes to maintain their performance?

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

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

What are the recommended blocking agents and conditions for minimizing background noise when using Invitrogen Low Fluorescence PVDF Membranes?

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.

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

Can Invitrogen Low Fluorescence PVDF Membranes be used for both chemiluminescent and fluorescent detection methods?

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

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

What is the optimal protein loading capacity for Invitrogen Low Fluorescence PVDF Membranes?

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.

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

How should I prepare and handle Invitrogen Low Fluorescence PVDF Membranes before use in western blotting?

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.

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

What is the pore size of Invitrogen Low Fluorescence PVDF Membranes?

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.

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

Are Invitrogen Low Fluorescence PVDF Membranes compatible with all types of fluorescent dyes and detection systems?

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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How does low background autofluorescence improve the quality of fluorescent western blotting results?

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.

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

How do Invitrogen Low Fluorescence PVDF Membranes compare to other types of membranes, such as nitrocellulose, in terms of performance and application?

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.

What are the advantages of using Invitrogen Low Fluorescence PVDF Membranes over regular PVDF membranes in western blotting?

Most standard PVDF membranes have high autofluorescence in fluorescent channels outside of the IR range. Invitrogen Low Fluorescence PVDF Membranes exhibit low background in all channels, allowing greater multiplexing using a wider range of bright fluorescent dyes and secondary antibodies. They exhibit low background even when excited with UV or 488 nm light sources, making them excellent for fluorescent total protein normalization methods.

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