After staining an IEF gel, what are reasons why the protein samples do not appear but the IEF protein standard (marker) does?
1) If the protein has a pI greater that 8.5, it may be able to be resolved on a pH 3-10 gel.
2) The protein may be insoluble.
3) There may be protein loss if the fixative is too weak in the fixation step prior to staining.
4) Your sample load may be too low.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Can I use your Protein standards for native gel applications?
We only offer one protein standard for native gel electrophoresis - NativeMark Unstained Protein Standard (Cat. No. LC0725) which is a ready-to-use protein marker for the estimation of the molecular weight of proteins using native gel electrophoresis with Tris-Glycine or NuPAGE Invitrogen Tris-Acetate Gels. It contains 8 proteins in the range of ~20-1200 kDa that can be visualized by Coomassie or silver staining.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
What are the recommended protocols for preparing Invitrogen protein standards to load on a gel?
Invitrogen protein standards do not require any additional preparation. Thaw protein standards at room temperature, vortex to mix well, and load.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting 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.
