Stocking western blot supplies and western blot reagents: choosing the western blot membrane or transfer membrane western blot chemistry for the target, which western blot blocking buffer or blocking buffer western blot work needs, why western blot paper thickness changes transfer more than the voltage does, and what a worn western blot cassette or western blot transfer cassette is doing to your uneven lanes

Choosing the consumables

1. Choose the membrane chemistry for the detection and the target. Nitrocellulose binds well, has low background and is fragile. Polyvinylidene fluoride is mechanically robust, binds more protein, requires methanol wetting and is the usual choice for fluorescent detection and for stripping and reprobing.
2. Pick the pore size from the protein size. A nought point four five micrometre pore suits most proteins; a nought point two micrometre membrane retains small proteins and peptides that otherwise pass straight through. A small target lost in transfer is very often a pore size choice nobody revisited.
3. Use thick blotting paper, cut exactly to the gel. Blotting paper holds the buffer reservoir that carries the current through the sandwich. Thin or unevenly cut paper gives uneven current and patchy transfer, and paper larger than the gel lets current bypass it entirely at the edges.
4. Retire cassettes and pads when they stop compressing. Foam pads compress permanently with use and a cassette that no longer applies even pressure gives exactly the patchy transfer people blame on the gel. Date the pads and replace them; they are cheap and they are consumables.
5. Hold the stock that stops work. Membrane, thick paper, pads, cassettes and blocking reagent for a fortnight. None is expensive and each of them has stopped a blot on a Friday afternoon somewhere.

Proving the transfer happened before you blame the antibody

A reversible total protein stain on the membrane after transfer, photographed and kept, answers the question that otherwise takes a week of antibody troubleshooting. It also gives a total protein normalisation better than most housekeeping proteins.

A prestained ladder visible on the membrane is the quick version of the same check and should be marked in pen immediately, because several dyes fade during blocking.

Wet, semi-dry and dry systems

Wet transfer is slow, gentle and the most reliable for large proteins. Semi-dry is much faster and can dry out on long transfers. Dry systems are fastest and use proprietary consumables, which is a lock-in worth pricing over five years.

Whichever is used, keep one method and one set of consumables for a study. Comparing blots transferred by different systems introduces a variable nobody records.

Blocking and buffers

Milk is cheap and interferes with phospho-specific antibodies; bovine serum albumin is the usual alternative and costs more. Test both for a new antibody rather than inheriting a choice.

Prepare buffers fresh and label them. A transfer that failed because the buffer was last week's, with the methanol evaporated, is a recurring and entirely preventable waste of a day.

Common questions

Nitrocellulose or PVDF?

Nitrocellulose for low background and straightforward chemiluminescence. PVDF for mechanical robustness, higher binding capacity, fluorescent detection and any blot you intend to strip and reprobe. PVDF must be wetted in methanol first.

Which pore size should I use?

0.45 micrometre for most proteins, 0.2 micrometre for small proteins and peptides. If a small target is transferring poorly, the pore size is the first thing to change.

Does western blot paper really matter?

Yes. It is the buffer reservoir that carries current through the sandwich, so thickness and an exact cut to the gel size decide how evenly the transfer happens. Paper overhanging the gel lets current bypass the sandwich.

How do I know a western blot transfer cassette is worn?

Patchy transfer that moves with the cassette rather than with the gel, and pads that no longer spring back. Date the pads when they are first used and replace them on that basis rather than waiting for a bad blot.

Browse by service class

• Compare Gene synthesis on advertised prices