Western blot transfer apparatus and blotting consumables: getting protein onto the membrane

Controlling the blot

1. Match the transfer mode to the protein size. Tank transfer is slow, gentle and best for very large proteins. Semi dry and cassette systems are fast and can drive small proteins through the membrane. Choose by the size range on the gel rather than by convenience.
2. Verify transfer before probing. A reversible total protein stain on the membrane, photographed, shows whether transfer worked and how evenly. It takes minutes and it prevents a day spent troubleshooting the antibody.
3. Choose the gel chemistry for the range. Low molecular weight proteins and peptides resolve poorly on a standard system and need a different buffer chemistry. Using the wrong gel puts the target in the dye front.
4. Fix blocking against the target class. Milk based blockers contain phosphoproteins and defeat phosphospecific antibodies. Protein free blockers suit those. The blocker is part of the method and a change is a method change.
5. Use total protein for normalisation. A stained membrane image gives a loading measure that does not depend on a housekeeping protein remaining constant under the treatment. It is now the expected normalisation for quantitative blots.
6. Know what an alternative platform replaces. Capillary and bead based systems automate the separation and detection and change the sample requirement and the dynamic range. They replace the labour, not the need to validate the antibody.

Stain the membrane, always

A reversible total protein stain immediately after transfer is the single most useful habit in blotting. It shows whether transfer occurred, whether it was even across the membrane, and whether loading was equal, before any antibody is spent.

It also provides the normalisation image that a quantitative blot needs, so it costs nothing extra.

Consistency comes from the handling

Uneven washing, membranes drying at the edges and variable incubation times cause more blotchy blots than any reagent. Incubation boxes, a timer and a fixed volume per membrane fix most of it.

Where blotting is a core activity, spend on handling before spending on a better imager. Consistent membranes photographed adequately beat inconsistent ones photographed beautifully.

BSA for western blot blocking, and where it goes

Bovine serum albumin appears twice in a blot and the two uses are different. As a blocking agent it coats the membrane, and it is the right choice where milk's own phosphoproteins or biotin would interfere, which means every phospho-specific antibody and every streptavidin detection. As a carrier in the antibody dilution it lowers non-specific binding. The grade matters in both: a fraction V albumin carries immunoglobulins that a secondary antibody will find, so a blot with a speckled background is often the albumin rather than the antibody.

transfer membrane western blot work needs, and choosing it

Two membranes carry almost all blots. Nitrocellulose binds protein well, gives low background and is brittle once dry, and it suits most chemiluminescent detection. PVDF is tougher, binds more protein per square centimetre, survives stripping and reprobing, and needs methanol wetting before it will take buffer at all. Pore size follows the target: 0.45 micrometres as standard, 0.2 for proteins under about 20 kilodaltons, which otherwise pass through. Both are consumables whose lot can change a background, which is why a troubleshooting log records the membrane.

A western blot membrane pvdf choice, and its handling

PVDF is hydrophobic, so it is wetted in methanol and then equilibrated in transfer buffer before the sandwich is assembled; a dry patch is a blank area on the film. Its higher binding capacity gives strong signals and also holds blocking agent and antibody more tenaciously, so washes are longer and background is higher on a low-quality blocker. In return it survives drying, storage and stripping for a second probe, which is why a precious sample goes onto PVDF and a routine blot onto nitrocellulose.

A horseradish peroxidase conjugated secondary antibody and its substrate

A horseradish peroxidase conjugated secondary antibody is chosen with its substrate and its exposure in mind, since the signal is enzymatic and accumulates, so a quantitative comparison needs an exposure inside the linear range. Azide in any buffer poisons the enzyme. The conjugate ratio decides the balance between sensitivity and background more than the dilution does.

A horseradish peroxidase secondary antibody in a tissue section

A horseradish peroxidase secondary antibody on tissue needs endogenous peroxidase blocked first, which is why blood rich sections stain brown with no primary at all, and the chromogen decides whether the result survives an alcohol dehydration. Development is stopped by eye against a control slide rather than by a fixed clock.

An anti-phosphotyrosine antibody and what it can screen

An anti-phosphotyrosine antibody recognises the modified residue across many sequence contexts, which makes it a screening and enrichment reagent rather than a site specific one, and its context bias means an enrichment with it is not unbiased. A phosphatase treated duplicate is the specificity control, and inhibitors in the lysis buffer are what keep the signal present at all.

Common questions

Why is my large protein missing?

It did not transfer. Very large proteins need longer, colder tank transfer, sometimes with methanol reduced and a low percentage gel. Stain the membrane and the gel afterwards to see where the protein went.

Why is my small protein missing?

It probably went through the membrane. Use a smaller pore size, shorten the transfer and add methanol, and confirm with a membrane stain.

Should I normalise to a housekeeping protein?

Total protein staining is safer, because housekeeping proteins change with treatment, confluence and stress more often than people assume. Show the total protein image with the blot.

Are western blot kits worth it?

For a standard workflow, they give consistency and remove preparation steps. For unusual targets or optimisation work, assembling the components gives the control that troubleshooting needs.

Browse by service class

• Compare Gene synthesis on advertised prices