| Q. |
What blotting membranes are compatible with SNAP i.d. system? |
| A. |
All standard membranes are compatible. For optimal performance, Millipore recommend Immobilon-P membrane for chemiluminescent detection,
or Immobilon-FL membrane for fluorescent detection |
| Q. |
What concentration of antibody should I use for an antibody that has never been tested in immunoblotting? |
| A. |
The antibody will require optimization testing just as it would for traditional western blot testing.
Millipore recommend starting at a high concentration. Refer to supplier's recommended concentration for western blotting,
then use 3- to 5-times the concentration in 1/3 to 1/5 the volume for SNAP i.d. immunodetection. |
| Q. |
Which blot holder is recommended for antibody optimization? |
| A. |
Either the double or triple well blot holder works well for optimization. The three well holder allows testing of up to
six different antibody concentrations at one time (3 antibodies per blot holder). |
| Q. |
How can I obtain even distribution of the antibody through the blot holder? |
| A. |
Make sure to include 0.1% Tween 20 surfactant in both the blocking and antibody solutions, as it reduces surface
tension and facilitates distribution. Applying the antibody solution evenly across the blot holder surface is essential
for optimal performance. The solution must cover the entire surface. If necessary, increase the antibody volume
slightly to ensure even coverage. |
| Q. |
Can an antibody be incubated longer than 10 minutes using the SNAP i.d. system? |
| A. |
Yes, however, longer incubation periods may increase background signal for some antibodies. |
| Q. |
Will my membrane dry out during the 10-minute incubation period? |
| A. |
No, the membrane will remain wet, even if the blot holder looks empty or dry. |
| Q. |
Can the primary antibody be incubated overnight in the SNAP i.d. system? |
| A. |
The protocol for the SNAP i.d system is designed to enable rapid immunodetection; overnight antibody incubation
should not be necessary. However, if overnight incubation is desired, Millipore recommend performing this incubation at 4°C.
Prolonged incubation may result in drying of the membrane and/or increased background. |
| Q. |
What will happen if my lab vacuum source is too high? |
| A. |
The SNAP i.d. system has an internal vacuum regulator that controls the amount of vacuum applied to the blot holder. |
| Q. |
What will happen if my lab vacuum source is too low? |
| A. |
The flow rate may be inconsistent, resulting in high background. It is essential that a minimum of 4" Hg be applied to the system. |
| Q. |
Why is the thin blot spacer required on top of the blot? |
| A. |
The blot spacer is required to keep the blot from contacting the grid of the blot holder. It ensures the even distribution of reagents to the blot. |
| Q. |
Can different proteins be detected at the same time when using a double or triple well device? |
| A. |
Yes, each well is completely independent thus different primary or secondary antibodies can be used in adjacent wells. |
| Q. |
Can the double or triple well blot holder be used when I only have one blot to test? |
| A. |
Yes, however, be sure to wet the unused wells with Milli-Q water at the beginning of the procedure. No subsequent reagents need to be added to the unused wells. |
| Q. |
Can both sides of the system be used independently? |
| A. |
Yes, each blot holder is controlled independently by the knob on its side of the system. |
| Q. |
Can I re-use a blot holder? |
| A. |
Re-use of blot holders is not recommended due to the risk of sample cross contamination and the potential for clogging.
However, if one of the wells of a double or triple blot holder has been wet only with Milli-Q water, that well can be used
in subsequent assays. Long term storage is not recommended in this situation. |
| Q. |
Can Western blots be stripped and reprobed using the SNAP i.d. system? |
| A. |
The SNAP i.d. system is intended for immunodetection only. Blots that have been stripped outside of the system following
a standard protocol can be reprobed in the SNAP i.d. system starting with the blocking step. |
| Q. |
Can I recover and re-use antibodies used with the SNAP i.d. system? |
| A. |
Yes, refer to Antibody Collection Tray Instructions |
| Q. |
Can the SNAP i.d. system be used right after blot transfer? |
| A. |
Yes, as long as the blot is still wet, place the blot in the blot holder and proceed with the Immunodetection Protocol. |
| Q. |
Are there alternative ways of wetting the blot holder? |
| A. |
Yes, there are several. You can place the closed blot holder in the SNAP i.d. system, fill the wells with Milli-Q water,
and filter to dryness. Alternatively, the opened blot holder can be submerged into a tray of Milli-Q water. The excess water should be removed. |
