How can you improve the transfer efficiency of the BenchMark protein ladder when Western blotting onto a PVDF membrane?
There are two factors to consider - poor transfer and the ladder passing through the membrane during the transfer.
For poor transfer onto membrane, consider:
-The percent acrylamide should be 8% to get rapid, more complete transfer of high-molecular weight proteins.
-Increase voltage, current, or length of time for transfer.
-For transfer to PVDF, omit the SDS from the transfer buffer. Addition of SDS (or use of old buffer that may have absorbed SDS leached in from the gel) will cause the proteins to bind less efficiently to PVDF membranes because it inhibits the hydrophobic interaction between the membrane and the protein.
If the problem is the protein staying in the gel, consider any of the following:
-Increase the SDS concentration to 0.1% (but use nitrocellulose).
-Eliminate the methanol in the buffer.
-Reduce the acrylamide percentage.
-Transfer longer.
If the ladder goes through membrane during transfer:
-Decrease voltage, transfer time.
-Check concentration of SDS and methanol. Too much SDS can prevent binding to membrane. Alcohol enhances hydrophobic binding to membrane; not enough alcohol may prevent binding.
-Use a 0.2 mm pore size of nitrocellulose.
-Check gel percentage; smaller proteins will pass through membranes more easily.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
What are the storage conditions and shelf life for the BenchMark Protein Ladder (Cat. No. 10747012)?
The BenchMark Protein Ladder is stable for six months if used and stored as recommended (-20 degrees C, avoid repeated freezing and thawing). We recommend aliquoting the ladder and storing it at -20 degrees C. Aliquots, once opened, should be stored at 4 degrees C.
Find additional tips, troubleshooting help, and resources within our Protein Standards and Ladders Support Center.
I used one of your protein standards for a western transfer and noticed that some of the lower-molecular weight protein bands passed through the membrane. How can I resolve this issue?
- Decrease voltage, current or length of transfer time
- Make sure that the methanol concentration in the transfer buffer is proper; use a methanol concentration of 10-20% methanol removes the SDS from SDS-protein complexes and improves the binding of protein to the membrane.
- Make sure that the SDS concentration (if added) in the transfer buffer is proper, don't use more than 0.02-0.04% SDS. Using too much SDS can prevent binding of proteins to the membrane.
- Check the pore size of the membrane and the size of the target protein. Proteins smaller than 10 kDa will easily pass through a 0.45 µm pore size membrane. If proteins smaller than 10 kDa are of interest, it would be better to use a 0.2 µm pore size membrane.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
I used one of your protein standards for a western transfer and noticed that some of the higher-molecular weight bands transferred very poorly to the membrane. Can you offer some tips?
- Increase voltage, current or length of time for transfer
- SDS in the gel and in the SDS-protein complexes promotes elution of the protein from the gels but inhibits binding of the protein to membranes. This inhibition is higher for nitrocellulose than for PVDF. For proteins that are difficult to elute from the gel such as large molecular weight proteins, a small amount of SDS may be added to the transfer buffer to improve transfer. We recommend pre-equilibrating the gel in 2X Transfer buffer (without methanol) containing 0.02-0.04% SDS for 10 minutes before assembling the sandwich and then transferring using 1X transfer buffer containing 10% methanol and 0.01%SDS.
- Methanol removes the SDS from SDS-protein complexes and improves the binding of protein to the membrane, but has some negative effects on the gel itself, leading to a decrease in transfer efficiency. It may cause a reduction in pore size, precipitation of some proteins, and some basic proteins to become positively charged or neutral. Make sure that the methanol concentration in the transfer buffer is not more than 10-20% and that high-quality, analytical grade methanol is used.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
I used one of your pre-stained standards on a Tris-Glycine gel and noticed that the molecular weights of the proteins were different than on a NuPAGE Bis-Tris gel. What is the reason for this?
Pre-stained standards have a dye that is covalently bound to each protein that will result in the standard migrating differently in different buffer systems (i.e., different gels). As a result, using a pre-stained standard for molecular weight estimation will only give the apparent molecular weight of the protein. Pre-stained standards may be used for molecular weight approximation, confirming gel migration and estimating blotting efficiency but for accurate molecular weight estimation, an unstained standard should be used.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
