Optiblot - Frequently Asked Questions

Optiblot Gels and Buffers

  1. How many gels can be run with a 500ml bottle of Run Buffer?
  2. Are Optiblot Run Buffers Tris-MOPS or Tris-Tricine buffers?
  3. Which tanks are compatible with Optiblot precast gels?
  4. What are the running conditions for Optiblot gels and buffers?
  5. Do I need to use Optiblot buffers?
  6. Can I make Optiblot buffers in my lab?
  7. How long can I store Optiblot gels for?
  8. I received my gels at room temperature is this okay?
  9. How thick is an Optiblot pre-cast gel?
  10. Can I use a standard Laemmli sample buffer to prepare samples for loading to Optiblot gels?
  11. Are gradient gels available in the Optiblot range?
  12. How many wells do Optiblot gels contain?
  13. What is the pore size of the PVDF membranes (ab133411)?

Optiblot Blue

  1. Do I need to do anything prior to staining my gel?
  2. How long does Optiblot Blue take?
  3. Do I still need to de-stain my gel?
  4. How do I dispose of Optiblot Blue?
  5. Will my gels over-stain?
  6. What is the sensitivity of Optiblot Blue?
  7. Is Optiblot Blue compatible with mass spectrometry (MS) and Edman sequencing?
  8. Can Optiblot Blue be used instead of a traditional coomassie stain with other acrylamide gels?
  9. Is Optiblot Blue a R250 or G250 based stain?

Optiblot Bradford Reagent

  1. Can I use Optiblot Bradford Reagent if my sample contains detergents?
  2. Which standards should I use for my standard curve?

Optiblot ECL

  1. How do the Optiblot ECL reagents compare to similar ECL reagents?
  2. What is the recommended exposure time to film when staining membranes with Optiblot ECL reagents?
  3. Are Optiblot ECL reagents compatible with fluorescent imagers (eg. Typhoon)?
  4. Can the Luminol membrane pen ab166858 be used to label membranes to be exposed for extended time periods?

Optiblot Gels and Buffers

1. How many gels can be run with a 500ml bottle of Run Buffer?

Optiblot Run Buffer is 20x concentrated and 500ml is sufficient for a full box of gels (i.e. 40ml Run Buffer per gel).

2. Are Optiblot Run Buffers Tris-MOPS or Tris-Tricine buffers?

Optiblot Reducing SDS Run Buffer is a reducing Tris-MOPS buffer system similar to conventional MES (excellent for the separation of low molecular weight proteins). Optiblot SDS Run Buffer is a non-reducing Tris-tricine buffer system providing similar separation to MOPS(increased separation of high molecular weight proteins.

3. Which tanks are compatible with Optiblot precast gels?

Tank system 10 x 10 Optiblot gel 8 x 10 Optiblot gel
Bio-Rad Mini-PROTEAN No Yes
Invitrogen XCell II Yes No
Invitrogen XCell Surelock Yes No
Thermo Scientific Owl P82 Yes No
CBS Scientific Quadra Combo Yes Yes

4. What are the running conditions for Optiblot gels and buffers?

Voltage 180v
Start current 90mA/gel
Ending current 40mA/gel
Run time 40-55 minutes

5. Do I need to use Optiblot buffers?

Yes, Optiblot buffers must be used with Optiblot gels to achieve the best results. Other buffer systems will result in poor electrophoresis.

6. Can I make Optiblot buffers in my lab?

Yes, buffer recipes are available to download.

7. How long can I store Optiblot gels for?

Optiblot gels can be stored for approximately 6 months at room temperature (RT), for longer term storage 4°C is recommended. Avoid freezing as this is likely to damage gels.

8. I received my gels at room temperature is this okay?

Yes, Optiblot gels are shipped at room temperature to avoid potential freezing.

9. How thick is an Optiblot pre-cast gel?

Optiblot gels are 1mm thick

10. Can I use a standard Laemmli sample buffer to prepare samples for loading to Optiblot gels?

It is not recommended. For optimal results, we recommend using Optiblot LDS Sample Buffer (4X) (ab119196).

11. Are gradient gels available in the Optiblot range?

Yes. Gradient gels include 4-8% and 4-20% gradient gels. Please find further details here.

12. How many wells do Optiblot gels contain?

Optiblot gels contain either 12 or 17 wells. Please find further details here.

13. What is the pore size of the PVDF membranes (ab133411)?

The pore size of this PVDF membrane is 0.45µm

Optiblot Blue

1. Do I need to do anything prior to staining my gel?

No, simply remove the gel from the cassette and place directly in the stain.

2. How long does Optiblot Blue take?

You can expect to see the strongest bands appear after around 5 minutes with all bands being visible after 15minutes. Total saturation of Optiblot Blue occurs after approximately 1 hour.

3. Do I still need to de-stain my gel?

No, Optiblot Blue has been formulated not to bind to the SDS or Acrylamide in the gel, providing a clear background.

4. How do I dispose of Optiblot Blue?

Optiblot Blue is non-toxic and can typically be disposed of in the sink. However, always check your local regulations.

5. Will my gels over-stain?

No, Optiblot Blue will not cause gels to be over-stained, even if left submerged for days.

6. What is the sensitivity of Optiblot Blue?

Optiblot Blue can clearly visualize a 5 ng band of BSA, however, this may not be the case for your protein and is also dependent on basicity - the more basic your protein the better the sensitivity. Typically, staining is in the range of 5 to 25 ng protein per band.

7. Is Optiblot Blue compatible with mass spectrometry (MS) and Edman sequencing?

Yes

8. Can Optiblot Blue be used instead of a traditional coomassie stain with other acrylamide gels?

OptiBlot Blue is a ready-to-use Coomassie stain that is optimal for obtaining well defined protein bands in a single step. It can be replaced by traditional Coomassie, and be used with any electrophoretic gel.

9. Is Optiblot Blue a R250 or G250 based stain?

Optiblot Blue is a G250 based stain.

Optiblot Bradford Reagent

1. Can I use Optiblot Bradford Reagent if my sample contains detergents?

Yes, Optiblot Bradford Reagent can resist the effects of detergents which normally influence standard Bradford assays.

2. Which standards should I use for my standard curve?

The Bradford assay is affected by the basicity and hydrophobicity of the protein. Typically, proteins have a basic amino acid composition of 10 to 17 mol% and you should try to use a standard as close to the mol% of basic amino acids as your unknown protein. Some good standards are shown below.

Standard Protein Mol% positive residues
Hen egg white lysozyme 13.9
Bovine serum albumin (BSA) 16.5
Bovine Immunoglobulin (IgG) 11.3
Bovine beta lactoglobulin 11.8

Optiblot ECL

1. How do the Optiblot ECL reagents compare to similar ECL reagents?

Optiblot ECL Detect (ab133406) can be used to detect expression of typical protein levels.
Optiblot ECL Max Detect (ab133408) can be used to detect relatively small amounts of protein.
Optiblot ECL Ultra Detect (ab133409) is one of the most sensitive ECL reagents on the market and can be used to detect extremely small levels of protein.

2. What is the recommended exposure time to film when staining membranes with Optiblot ECL reagents?

When optimizing exposure for your western blotting, we recommend initially trying 30 second, 2 minute and 5 minute exposures. However the blot can be imaged for several hours if necessary.

3. Are Optiblot ECL reagents compatible with fluorescent imagers (eg. Typhoon)?

Optiblot ECL Ultra (ab133409) produces a chemifluorescent signal that can be detected with a fluorescence imaging system using appropriate excitation and emission settings. Other Optiblot ECL reagents are not compatible with fluorescent imagers. However our Fluorescent western blot kit (ab133410) IS fully compatible with non-IR imagers like Typhoon, etc.

4. Can the Luminol membrane pen (ab166858) be used to label membranes to be exposed for extended time periods?

When the membrane needs to be exposed for a long time, we suggest drawing a weaker band as far as possible from the lanes on the membrane in order to avoid interference.

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