Anti-BRCA2抗体(ab9143)

Thanks for your inquiry. I have copied below some details on transferring large proteins. Detailed instructions for the transfer process can be found on the websites of the manufacturers of transfer apparatus, and will vary depending on the system. The principle is the same in each case, though. Just as proteins with an electrical charge (provided by the SDS bound to them) can be induced to travel through a gel in an electrical field, so can the proteins be transferred in an electrical field from the gel onto a sturdy support, a membrane that “blots” the proteins from the gel. (Early methods relied on diffusion; blotting in an electrical field is now standard). Transfer can be done in wet or semi-dry conditions. Semi-dry transfer is generally faster but wet transfer is a less prone to failure due to drying of the membrane and is especially recommended for large proteins, >100 kD. For both kinds of transfer, the membrane is placed next to the gel. The two are sandwiched between absorbent materials, and the sandwich is clamped between solid supports to maintain tight contact between the gel and membrane. In wet transfer, the gel and membrane are sandwiched between sponge and paper (sponge/paper/gel/membrane/paper/sponge) and all are clamped tightly together after ensuring no air bubbles have formed between the gel and membrane. The sandwich is submerged in transfer buffer to which an electrical field is applied. The negatively-charged proteins travel towards the positively-charged electrode, but the membrane stops them, binds them, and prevents them from continuing on. A standard buffer for wet transfer is the same as the 1X Tris-glycine buffer used for the migration/running buffer without SDS but with the addition of methanol to a final concentration of 20%. For proteins larger than 80 kD, it is recommended that SDS is included at a final concentration of 0.1%. Two types of membranes are available: nitrocellulose and PVDF (positively-charged nylon). The choice is personal and both work very well. PVDF membranes require careful pre-treatment: cut the membrane to the appropriate size then soak it in methanol for 1-2 min. Incubate in ice cold transfer buffer for 5 minutes. The gel needs to equilibrate for 3-5 minutes in ice cold transfer buffer. Failure to do so will cause shrinking while transferring, and a distorted pattern of transfer. Note on transfer of large and small proteins The balance of SDS and methanol in the transfer buffer, protein size, and gel percentage can affect transfer efficiency. The following modifications will encourage efficient transfer. Large proteins (>100 kD) For large proteins, transfer out of the gel may be very slow, just as they run slowly within the gel during separation. If blotting a large protein, be sure to run your samples in a low-concentration gel, 8% or less. These will be very fragile, so handle carefully. Large proteins will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0.1% in the transfer buffer will discourage this. Methanol tends to remove SDS from proteins, so reducing the methanol percentage to 10% or less will also guard against precipitation. Lowering methanol in the transfer buffer also promotes swelling of the gel, allowing large proteins to transfer more easily. Methanol is only necessary if using nitrocellulose. If using PVDF, methanol can be removed from the transfer buffer altogether, and is only needed to activate the PVDF before assembling the gel/membrane sandwich. Choose wet transfer overnight at 4°C instead of semi-dry transfer. I hope you find this information useful. Please feel free to contact us if you require further assistance.

Thank you for your enquiry and your interest in our products. We would like to confirm that the two product are the same and from the same originator. The only difference is that ab1292 is Rabbit polyclonal to BRCA2 (Agarose) suitable for only IP application.