#Western blot sandwich free#

The universal Transfer-buffer is: 25 mM Tris, 192 mM glycine, 10-20% MeOH, w/o SDS (use up to 0.1% SDS for proteins > 80 kDa). Only for Semi-dry systems the pH should be below 8.5 for not damaging the graphite electrodes. Several recipies exits - indicating that the exact composition is not too important. In the CBL we have the Wet-blot 'Mini Trans-Blot'-system ( #170-3935) which is compatible with the SDS-PAGE Mini-Protean Tetra Cell ( #165-8001) from Biorad. of 10% (instead of 20%) can be a good choice (or PVDF-membranes can be used w/o MeOH).įor small proteins the SDS can hinder the binding of the proteins on the membrane - thus it may good to remove the SDS completely from the Transfer-buffer and keep the MeOH at 20%.īefore the run, gel and membrane should be equilibrated for 15 min in ice-cold Transfer-buffer (otherwise shrinking will occure resulting in a distorted transfer-pattern).ĭuring the run the ice-cold buffer should be stirred (add a magnetic bar and place system onto a magnetic stirrer) to maintain uniform temperature and thus conductivity. MeOH tends to remove SDS from the proteins, thus a final MeOH conc.

Generally, the transfer of large proteins (> 100 kDa) is slow, they tend to precipitate within the gel, so adding 0.1% SDS is recommended. The Wet or Tank blot has less harsh conditions and is util for large proteins (> 100 kDa), but requires bigger quantities of transfer buffer and is slower than the Semi-dry blot on the other side, the Semi-dry blot generates more heat, which can damage the protein (detection) and colour the membrane. In any case a sandwich is formed from: cathode (-) - (sponge) - paper - gel - membrane - paper - (sponge) - anode (+). Principally 3 types for blotting are possible: Wet, Semi-dry and Dry. In the CBL we use Whatman Protran NC-membranes with 0.45 µm pore size, precutted (7 x 8.5 cm), and prepackaged into a sandwich with 2 sheets of 3MM blotting paper portein binding capacity is stated to be 80 µg/cm 2 ( BA85/3MM, #10485374). For small proteins ( 100 kDa) SDS can be added to the Transfer-buffer to increase the mobility (but it decreases the binding on NC). The Transfer-buffer requires MeOH for better binding characteristics. To activate NC-membranes, place them *on* the surface of the Transfer-buffer, wait until they got grey, then submerge them into the buffer. With 100 µg protein per lane, each band has less than 10 µg, thus the protein binding capacity usually is more than enough. NC-membranes have a high protein binding capacity (80-100 µg/cm 2), non-specific protein binding sites are easily blocked (low background) and are much cheaper than PVDF-membranes. MeOH can be completely removed from the Transfer-buffer ( e.g.for the transfer of large proteins) - but nevertheless, activate PVDF-membrane with MeOH.īe careful, NC-membranes are brittle when dry, thus supported NC-membranes are recommended. To activate the PVDF-membrane soak it for 1-2/ min in MeOH before use (do not let them dry afterwards or re-soak). PVDF-membranes have a very high protein binding capacity (170-200 µg/cm 2), thus it is more prone for higher background. This also reduces the moisture between gel and membrane for having better contact between both and thus better transfer. It is important not having air bubbles between the membrane and the gel: roll out the membrane e.g. The membrane once placed onto the Gel should not be moved further (protein can already adsorb). Also the gel should be equilibrated for 15 min in ice-cold Transfer-buffer (otherwise shrinking will occure resulting in a distorted transfer-pattern). The membranes as well as the filterpaper are pre-soaked with Transfer-buffer (PVDF-membranes are additionally activated with MeOH previously) for ~ 15 min. Make sure that gel, membrane and paper are from the same size: large overhangs can prevent efficient current and transfer of the proteins. Therefore, it is important not to touch those membranes with your fingers (use gloves!). The main characteristic of the membrane is that it adsorbs protein. The proteins, separated by a SDS-PAGE, are transfered onto a membrane applying an electric field.

#Western blot sandwich free#

TRANSFERENCE OF PROTEINS ON A MEMBRANE AND SPECIFIC DETECTION WITH ANTIBODIESġ) Transfer of the protein on the membrane (blotting)Ģ) Saturation of free binding site of the membrane (blocking)ģ) Specific detection of Your Favorite Protein (YFP) with antibodiesĪ nice animation was made by Lodish et al.