Fractionate or concentrate the sample using one or more of these techniques:.

Check concentration of protein samples (e.g., using Bradford or Lowry protein assays).

(see also Protein Band Appearance > No Bands)Īll bands appear very faint, or you see no signal with chemiluminescence or fluorescence detection.įor more hints on how to improve transfer and binding, see also Troubleshooting: Transfer in the Protein Blotting Guide

Make sure the membrane is fully immersed and agitated throughout incubations.

Not enough solution used during incubation and/or washing Transfer tank must contain sufficient buffer to entirely cover blot area

Completely fill transfer tank with buffer.

Ensure that warm membranes are not allowed to dry after transfer by shortening handling time

High-intensity or rapid transfer methods generate heat that may cause the membrane to dry while it is handled.

A completely wet PVDF membrane has a gray, translucent appearance

When using a hydrophobic PVDF membrane, it must be completely soaked in methanol prior to equilibration in aqueous transfer buffer.

Ensure that membranes are uniformly wet before transfer.

Membrane not uniformly wet before transfer Transfer efficiencies of 60 to 80% may be achievable for proteins between 14 and 116 kDa, longer transfer times are required to transfer higher molecular weight proteins.Some sections of the blot appear to have less protein on them. Methanol-free transfer buffers are recommended to reduce transfer time to 7 to 10 minutes. Electrotransfer is performed either at constant current (0.1 up to ~0.4 A) or voltage (10 to 25 V) for 10 to 60 minutes. Methanol may be included in the transfer buffer, but other organic solvents, including aromatic hydrocarbons, chlorinated hydrocarbons and acetone, should not be used to avoid damage to the semi-dry blotter.

One to four gels may be rapidly electroblotted to membranes. Likewise, it is key that the filter paper sheets and membrane are cut to the size of the gel. To maximize the current passing through the gel instead of around the gel, the amount of buffer available during transfer is limited to that contained in the sandwich, so it is helpful if the extra-thick filter paper (~3 mm thickness) and membrane are also sufficiently soaked in buffer. For this semi-dry transfer, it is very important that the gel is pre-equilibrated in transfer buffer. With increasing time, however, there is a risk of over-transfer (blew through) of the proteins through the membrane, especially for lower molecular weight (<30 kDa) proteins when using membranes with a larger pore size (0.45 µm).įor semi-dry protein transfer, the transfer sandwich is placed horizontally between two plate electrodes in a semi-dry transfer apparatus. The transfer efficiency improves with increased transfer time.

Transfer efficiencies of 80–100% are achievable for proteins between 14–116 kDa. A high field option exists for a single gel, which may bring transfer time down to as little as 30 minutes, but it requires the use of high voltage (up to 200 V) or high current (up to 1.6 A) and a cooling system to dissipate the tremendous heat produced. Transfers are typically performed with an ice pack and at 4☌ to mitigate the heat produced. Multiple gels may be electrotransferred in the standard field option, which is performed either at constant current (0.1 to 1 A) or voltage (5 to 30 V) from as little as 1 hour to overnight. The supported gel sandwich is placed vertically in a tank between stainless steel/platinum wire electrodes and filled with transfer buffer. The gel is then placed in the “transfer sandwich” (filter paper-gel-membrane-filter paper), cushioned by pads and pressed together by a support grid. When performing a wet transfer, the gel is first equilibrated in transfer buffer. SureLock Tandem Midi Gel Tank: capacity for up to 2 blot modules XCell SureLock Mini-Cell: capacity for up to 2 blot modules Mini Gel Tank: capacity for up to 2 Mini Blot Modules Pre-assembled Select transfer stacks: buffer not required 1 mini gel per blot module 1–2 blot modules per tankġ midi gel per blot module 1–2 blot modules per tank

Transfer western blot