MagicMark™ XP Western-Proteinstandard

The Positope control protein (Cat. No. R90050) is designed to be used as a positive control in western blotting for a variety of antibodies. The control protein is a 53 kDa highly purified recombinant protein that contains seven epitope tags including the Xpress, c-myc, V5, His(C-term), HisG(N-term), Thioredoxin, and GFP.

The MagicMark XP Western Protein Standard (Cat. No. LC5602) consists of peptides that contain an IgG binding domain. These peptides can be detected with your regular antibodies and provide a way for you to get good molecular weight estimations without having to rely on transferred pre-stained standards.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

MagicMark XP standards consist of a mixture of eight E. coli-expressed recombinant proteins ranging in size from 20 to 120 kDa. Each protein contains an IgG binding sequence from Protein G. This IgG binding site binds the primary or secondary antibody used to detect your target protein, allowing you to detect the MagicMark standard directly.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Here are some suggestions:

- Verify that the detection reagents are working well. Optimize the antibody concentration to obtain best results.
- Make sure that the amount of standard loaded on the gel is correct.
- Optimize the transfer conditions (current, voltage, transfer time).
- Enzyme-conjugated primary antibodies may not bind efficiently with the proteins in the MagicMark XP Western Protein Standard. We recommend using unconjugated primary antibody, followed by the addition of enzyme-conjugated secondary antibody.
Note: The Anti-myc-AP/HRP and Anti-V5-AP/HRP antibodies do not bind to MagicMark XP proteins.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

- 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.

- 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.