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SNAP i.d. Protein Detection System

Frequently Asked Questions About...
SNAP i.d. systemReagents
Immunodetection processAntibody recovery and reuse
 
Q. Which additional equipment or material do I need for the SNAP i.d. System?
  • You will need:
    a) Vacuum source (pump or other uniform vacuum source)
    b) Vacuum tubing to connect flask to vacuum source
    c) Filter to protect the pump or vacuum source (Millex®-FA50)
    d) Flask trap (1 or 4 L) with No 8 perforated stoppers (XX1004708)

    For a complete list and setup instructions refer to the SNAP i.d. User Guide.

Q. What kind of trap should I use with the SNAP i.d. System?
  • You can use a minimum 1L flask (XX1004705) or 4L (XX1004744) Millipore with No 8 perforated stoppers (XX1004708). You can also use any container compatible with vacuum.

Q. What type of vacuum source should I use with the SNAP i.d. System?
  • The system is compatible with most laboratory “house” vacuum sources or vacuum pumps. Any uniform vacuum source that can deliver a sustained minimum pressure of 8" Hg (270 millibar) is acceptable. However if you are recovering your antibody a minimum of 12” Hg (410 millibar) is required.

Q. What will happen if my lab vacuum source is too high?
  • 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?
  • The flow rate may be inconsistent, resulting in high background. It is essential that a minimum of 8” Hg (270 millibar) be applied to the system. For antibody collection, 12” Hg (410 millibar) is required.

Q. How often should I clean my SNAP i.d. System?
  • Millipore recommends that the SNAP i.d. System be flushed with Milli-Q® water after each use. This is accomplished by actuating the vacuum and adding Milli-Q water to the wells. Users should also make sure that no buffer is left on the gaskets. If necessary, the unit can also be rinsed under tap water.

Q. Can I use alcohol or other organic solvents to clean the SNAP i.d. System?
  • No, use only Milli-Q water for cleaning. The use of any solvent other than water may result in damage to the system.

Q. Can both sides of the system be used independently?
  • Yes, each blot holder is controlled independently by the knob on its side of the system.

Q. Are the SNAP i.d. System and the blot holders compatible with acids or bases?
  • The SNAP i.d. Protein Detection System is compatible with aqueous solutions and dilute acids and bases.

Q. Is the SNAP i.d. System compatible with fluorescent detection?
  • Yes, the SNAP i.d. System is compatible with all detection methodologies, including fluorescence. For further information, refer to the application note entitled: “Fluorescent Immunodetection Using the SNAP i.d. Protein Detection System” (AN1967EN00).

Q. Why sometimes after washing I found water at the bottom of the unit?
  • If the system wells are filled with water before the vacuum is applied, water will leak from the base. This will have no effect on the function of the unit. To avoid this, activate the vacuum before adding water used for washing. To clear away any water left within the system, simply activate the vacuum.

Q. Why do I sometimes hear noise when activating the vacuum?
  • This is caused by a resonance frequency of the internal vacuum regulator. If this occurs, adjust the input vacuum at its source until the noise stops.

Q. What solution should be used wet the blot holder?
  • Milli-Q-water

Q. What is the best way to wet the blot holder?
  • Place a new, closed blot holder in the SNAP i.d. System, fill the wells with Milli-Q water and activate the vacuum until the wells empty.
  • Open the blot holder and submerge it into a shallow tray of Milli-Q water. Be sure to remove any excess water before loading a blot into the blot holder. The blot roller included with the SNAP i.d. System can used to remove the excess water.
  • Open the blot holder and apply Milli-Q water directly from a squirt bottle or with a pipette.

Q. Are there alternative ways of wetting the blot holder?
  • 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.

Q. Can the double or triple well blot holder be used when I only have one blot to test?
  • Yes, however, be sure to wet the unused well with Milli-Q water at the beginning of the procedure. No subsequent reagents need to be added to the unused wells.

Q. Which blot holder is recommended for antibody optimization?
  • Either the double or triple well blot holder works well for optimization. The three well holder allows testing for up to six different antibody concentrations at one time (3 antibodies per blot holder) when using both sides of the system.

Q. Can I re-use a blot holder?
  • Re-use of blot holder 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 different proteins be detected at the same time when using a double or triple well device?
  • Yes, each well is completely independent thus different primary or secondary antibody can be used in adjacent wells.

Q. What volume of blocking/antibody solution and wash buffer is recommended for mini-gel size blot?

ReagentSingle WellDouble WellTriple Well
Blocking solution30 mL15 mL10 mL
Antibody3 mL1.5 mL1 mL
Wash bufer30 mL15 mL10 mL


Q. What is the minimum volume of antibody required to completely cover the blot holder surface area?
  • The minimum volume is 3 mL for single well, 1.5 mL for double well and 1 mL for triple well blot holder.

Q. Can Western blots be stripped and reprobed using the SNAP i.d. system?
  • 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. The blot holders do not snap closed. What should I do?
  • If the blot holder becomes difficult to snap closed, apply Milli-Q water to the catch and/or latch area of the blot holder. The blot holders will now snap closed.
 
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Immunodetection Process

Assembly of blot holder
Q. Is the orientation of the western blot within the blot holder important for immunodetection with the SNAP i.d. System?
  • Proper positioning of the membrane is critical for optimal performance. The protein side of the western blot (i.e., the side of the membrane that contacted the polyacrylamide gel) should always be in direct contact with the blot holder membrane (see diagrams in the User Guide). Blots placed with protein side away from the blot holder membrane will show significantly lower signal.

    Q. Why is the thin blot spacer required on top of the blot?
  • The blot spacer is required to keep the blot from contacting the grid of the blot holder. It ensures the even distribution of the reagents to the blot.

Q. Can I use 3MM paper in place of the blot spacer?
  • The blot spacer is made of low protein binding polypropylene material with dimensions optimized for use with the blot holders. Use of other types of spacers or supports is not recommended.

Q. What size blot can I process in SNAP i.d.?

Blot holderMaximum Western Blot
Single well7.5 x 8.4
Double well4.2 x 8.4
Triple well2.8 x 8.4

Q. Can I stack multiple blots in a blot holder well?
  • We do not recommend stacking blots within the blot holder as it will result in high signal variability.

Q. What blotting membranes are compatibles with SNAP i.d. System?
  • All standard (nitrocellulose and PVDF) membranes are compatible. For optimal performance we recommend Immobilon-P membrane for chemiluminescent detection or Immobilon- FL membrane for fluorescent detection.
    Q. Can the SNAP i.d. System be used right after blot transfer?
  • Yes, as long as the blot is still wet.
    Q. Is any danger of drying the blot if the vacuum is applied for too long?
    • No, even if you keep the vacuum running for several minutes the blot will not dry since some liquid always remains within the blot holder.

    Blocking

    Q. Can I use non-fat dry milk (NFDM) for blocking in the SNAP i.d. System?
    • Yes, NFDM is excellent blocking reagent for the SNAP i.d. Detection System. The optimal concentration of NFDM in SNAP i.d. is 0.5%.

    Q. I only have low-fat dry milk available. Can I use that with the SNAP i.d. System?
    • Yes, follow the same recommendations that are made for using non-fat dry milk.

    Q. Can I use NFDM at concentration lower than 0.5%?
    • Yes, we have found adequate blocking with high signal to noise ratios using concentrations of NFDM as low as 0.05%. See the application note entitled: “Key Steps for Successful Immunodetection Using the SNAP i.d. Protein Detection System” (AN1966EN00).

    Q. Why I am getting poor liquid flow through the blot holder?
    • Check for the following:
      -Millex®-FA50 has become wet. Replace the filter.
      -The concentration of NFDM is higher than 0.5%. Use the recommended concentration of NFDM.
      -You are re-using a blot holder. Use a new blot holder.
      -The vacuum source is too low. Increase the amount of input vacuum. If necessary, convert to a vacuum pump capable to sustain 12” Hg (410 millibar).

    Q. Is SNAP i.d. compatible with blocking reagents other than NFDM?

    Blocker CompatibleRecommended Concentration
    Non-fat/low fat dry milk
    yes, ≤ 0.5%
    0.5%
    Casein, N-Z-Amine AS (Sigma)
    yes, ≤ 5%
    1%
    Bovine Serum Albumin (BSA)
    yes, ≤ 5%
    1%
    PVP-40 (Polyvinylpyrrolidone)
    yes, ≤ 1%
    1%
    Immunoblot Blocking Reagent
    yes, ≤ 0.5%
    0.5%
    BLOT-QuickBlockerTM Reagent
    yes, ≤ 0.5%
    0.5%, pre-filter
    ChemiBLOCKERTM Reagent
    yes
    ≤ 50%
    SEA BLOCK Blocking Buffer (Pierce)
    yes
    undiluted
    SuperBlock® Blocking Buffer (Pierce)
    yes
    undiluted
    Li-Cor® Odyssey® Blocking Buffer
    yes
    undiluted
    Gelatin
    not compatible
    N/A
    Antibody addition

    Q. How much primary and secondary antibody should I add?
    • If you know the concentration used in standard immunodetection, use 3- to 5-fold higher concentration in 1/3 of the volume (3 mL for single well blot holder). Further optimization will likely be required. See application note entitled: “Triple Well Blot Holder as a Tool for 30-Minute Western Blotting Optimization with the SNAP i.d. Protein Detection System” (AN1965EN00).

    Q. What concentration of antibody should I use for an antibody that has never been tested in immunoblotting?
  • The antibody will require optimization testing just as it would for traditional western blot testing. We 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. What is the best approach to optimize an antibody for use with the SNAP i.d. System?
    • By using two triple well blot holders, six different concentrations of primary antibody can be evaluated in parallel. See application note entitled: “Triple Well Blot Holder as a Tool for 30-Minute Western Blotting Optimization with the SNAP i.d. Protein Detection System” (AN1965EN00).

    Q. If I have to leave the lab for longer than 10 minutes during the incubation period, will my experiment be damaged?
    • No, you can leave the SNAP i.d. unattended for periods longer than 10 minutes and the membrane will not dry out or affect the immunodetection. Antibody incubation periods of 30 minutes to 1 hour may slightly increase the signal and/or background.

    Q. Can the primary antibody be incubated overnight in the SNAP i.d. System?
    • 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 we recommend removing the blot from the blot holder after blocking and incubating the blot under the standard condition (e.g., standards antibody concentration in a rocking tray). The next morning re-assemble the blot in the blot holder and continue with the SNAP i.d. immunodetection.

    Q. How can I obtain even distribution of the antibody through the blot holder?
    • Maker sure to include 0.1% tween-20 surfactant in the blocking solution 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 min using the SNAP i.d. System?
    • Yes, however, longer incubation periods may increase background signal for some antibodies.

    Q. Will my membrane dry out during the 10-minute incubation period?
    • No the membrane will remain wet even if the blot holder looks empty or dry.

    Q. Can I perform the chemiluminescent detection in the blot holder?
    • This is not recommended as it may result in lower chemiluminescent signals.

    High background
    Q. How can I reduce the background of my blot?
    • High antibody concentration is usually the cause of high background
      -If there are many non-specific bands: Reduce the primary antibody concentration
      -If there is high background over the entire blot (not related to the bands): Reduce the concentration of the secondary antibody.
      -If there is an area with an overall high background: Washing was insufficient. Make sure to follow the recommendations in the user guide for washing.
      - If there are big, black spots around the desired band at high concentrations of sample: Reduce the sample concentration

    Q. Why I am getting a dark edge around my blot?
    • The blot spacer was not aligned properly over the blot or the blot was too large for the blot holder used.
    • If the blotting membrane is cut with dull scissors, the edges may become damaged or collapsed resulting in a dark edge effect.

    Q. Why I am getting some pin point spotting in random places on the blot or dark artifacts in the blot?
    • Pin point spotting can be related to bubbles or un-even wetting of the blot.
    • Larger dark artifacts in PVDF membrane sometimes are observed in places where the membrane was handled with the tweezers or that were inadequately wet with methanol. Do not handle PVDF membranes with tweezers until complete wet.
     
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    Reagents

    Q. Can I use PBST instead of TBST?
    • Yes.

    Q. What is the maximum concentration of Tween-20 surfactant that I can use in SNAP i.d.?
    • We have tested Tween-20 up to 1% with success.

    Q. What is the minimum concentration of Tween-20 surfactant that I can use with the SNAP i.d. System?
    • Tween-20 is important to ensure even distribution of the antibody. Use of Tween-20 at lower than 0.1% is not recommended.
     
    Antibody Recovery and Re-use

    Q. Can I recover and re-use antibodies used with the SNAP i.d. System?
    Q. How much of my antibody can I expect to recover using the SNAP i.d. Antibody Collection Trays?
    • Recovery is typically about 80%. It is very important to:
      - Place the antibody recovery trays in the correct position. In the case of the 2-well blot holder, the tabs of the tray should always face each other. See Antibody Collection Tray instructions for details.
      -Make sure the latch of the SNAP i.d. System is completely closed.
      -Wait a few extra seconds for the vacuum to pull the antibody through and collect in the tray.
      -Ensure that the vacuum pressure is at 12” Hg.

    Q. How many times can I re-use my antibodies after collection?
    • You will be able to re-use the antibody at least once.

    Q. Why do I need to add extra buffer when collecting my antibodies?
    • The device has a dead volume which needs to be displaced in order to maximize the collection of antibody.

    Q. The collected antibody is on a higher volume than I started. How much of that should I use to probe a second blot?
    • You need to apply the entire amount of collected antibody for optimal signal.



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