With your SuperSignal Molecular Weight Protein Ladder/ SuperSignal Enhanced MW Protein Ladder, I see dye front interference in fluorescent applications. What should I do?
This is likely due to the dye-front not run off the gel or removed from the blot. We recommend running the dye front off of the gel or removing it from the blot.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
I used your SuperSignal Molecular Weight Protein Ladder and did not see any bands after western detection. What could have happened?
Here are possible causes and solutions:
- Not enough volume loaded: Load more volume onto the gel
- Incomplete or poor transfer: Optimize transfer conditions
- Secondary antibody does not recognize marker proteins: Ensure appropriate secondary is used that binds to marker proteins
- Secondary antibody not enzyme-conjugated: Ensure appropriate secondary antibody used in system
- A mouse monoclonal primary antibody was used: Use a greater volume of the ladder or use SuperSignal Enhanced Molecular Weight Protein Ladder (Cat. No. 84786) for mouse primary antibodies
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
With your SuperSignal Molecular Weight Protein Ladder and SuperSignal Enhanced MW Protein Ladder, I did not get visible band separation after western detection. What could have happened?
Here are possible causes and solutions:
- Ladder was boiled: Discard boiled aliquot.
- Too much volume of ladder used: Load less volume of the ladder.
- Concentration of antibody is too high: Optimize antibody concentration
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 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.
