Filters

Condition A: Ready Filter™ with Xtalscint® solid scintillator

• Whole Cell/Homogenate Assays (e.g., receptor, cell proliferation)
  Ready Filter may be used to replace traditional glass fiber filters. Ready Filters have the sample retention characteristics of Whatman GF/B filters (1 micron cutoff). Use your routine procedure (prewetting/presoaking, harvesting) with the following modifications:
  1. The vacuum source should be a pump capable of delivering consistent suction of at least 25-inch (640 mm) Hg.
  2. After filtration, dry the filters, if possible, before placing or punching into counting vials.

  Note:
  When using a harvester, poor replicate variability is frequently the result of uneven buffer flow to individual filtration areas, resulting in variable filter blanks. To check this, filter liquid containing the same amount of radioactivity through all filtration areas using your normal procedure, then dry and count the filters. If variability of filter blanks is excessive, refer to your harvester manual for instructions on adjusting wash buffer flow rates.
   

• TCA Precipitates
  10% TCA may be used with Ready Filter without problems. After depositing the precipitates, remember to wash with 10% TCA to avoid solubilization. For suggestions on how to minimize self-absorption with Ready Filter, refer to the data sheet "Filtration Assays and Quench Resulting from Beta Particle Self-Absorption" in the Appendix.
  
  For further details on the use of Ready Filter consult the section on Xtalscint and Ready Filter and the instructions supplied with each box of Ready Filter.

Condition B: Cocktail Counting

• Sample capable of elution by cocktail
  1. Add to the filter either 8-10 mL Ready-Solv HP™ or Ready Safe™, or 4-5 mL of Ready Flow III™ or Ready Protein+™ and shake. The filter must face up.
  2. Let stand 30 minutes at room temperature. For Ready Safe, increase standing time to 2 hours.
  3. Count.

  Expected tritium efficiencies: 40-50% for Ready-Solv HP and 35-45% for Ready Flow III, Ready Protein+ and Ready Safe.

  Note:
  Quantitative recovery of counts in the filter may require prolonged (greater than 12 hours) cocktail extraction if the filter is not dried prior to cocktail addition.
   

• Sample not capable of elution by cocktail
  1. Add 0.4 mL of 0.1 N NaOH or KOH to the filter (face up) in a counting vial.
  2. Incubate one hour to overnight at room temperature.
  3. Add 4-10 mL Ready Protein+, Ready-Solv HP, Ready Flow III or Ready Safe.
  4. Count.

  Expected tritium efficiencies for Ready-Solv HP, Ready Protein+ and Ready Safe are 40-45%; for Ready Micro 34-39%.

  Warning:
  Unless the deposited sample is completely solubilized from the surface of the filter, beta particle self-absorption makes quench correction methods unreliable. Cocktails that do not contain emulsifiers should not be used for filter counting. For further details on filter counting with cocktails, refer to the article "Pitfalls for the Unwary in Liquid Scintillation Counting".

TCA Precipitates

1. Moisten 100 mg of dried TCA precipitate with 0.1-0.2 mL of water.
2. Rehydrate for 30 minutes.
3. Solubilize with 0.1 mL of 0.1M KOH or NaOH.
4. Incubate for 30 minutes at room temperature until solubilized
5. Add 10 mL Ready Protein+ or Ready Safe.
6. Shake well and count.

Expected efficiency for tritium is 35-45%.

TLC Scrapings

Condition A: Water-soluble sample

1. Add 1 mL of water to the scrapings.
2. Incubate 3-5 hours at 40ºC.
3. Add 8-10 mL of Ready Gel.
4. Shake and count.

Expected tritium counting efficiency is 37-43%.

Condition B: Samples not soluble in water

1. Add 1 mL of BTS-450 to the scrapings.
2. Incubate for 3-5 hours at 40ºC.
3. Add 8-10 mL of Ready Organic containing 7 mL/liter of glacial acetic acid¹.
4. Count.

Expected tritium counting efficiency is 47-53%.

Note:
1. See section on reducing chemiluminescence.

Polyacrylamide Gels with Bis-Acrylamide Cross-Linking

Condition A: Ready Filter (for ³²P and ¹²⁵I only)

1. Run and stain gel. Use usual procedure.
2. Cut out bands and place on Ready Filter circles (Xtalscint side up) in the bottom of 20 mL counting vials.
3. Count with a wide open window (or use the Xtalscint CPM/DPM option on LS 6000 scintillation counters).
4. For background subtraction, count an equal sized piece of gel from an area without stained bands.
5. The Ready Filters may be reused without contamination.

Note:
This procedure is taken from Pillion, D.J., Kim, S.J. and Meezan, E. Direct Scintillation Counting of ³²P and ¹²⁵I Incorporated into Proteins Separated by Polyacrylamide Gel Electrophoresis. See also Beckman Coulter Technical Bulletin T-1696-NUC-90-2.

Condition B: For All Beta Emitters

1. Place the gel slice in a vial. Add 0.2 mL of 60% perchloric acid and swirl.
2. Add 0.4 mL of 30% hydrogen peroxide and swirl again. Cap the vial.
3. Incubate at 70-80ºC for 30-60 minutes with gentle agitation if possible.
4. Cool the sample and add 7 mL of Ready Protein+ or Ready Safe. A clear solution should result.
5. Count.

Note:
This procedure is taken from "Determination of Several Isotopes in Tissues by Wet Oxidation" (Chapter 22; Authors: D.T. Mahin and R.T. Lofberg) in "The Current Status of Liquid Scintillation Counting" (ed.: E.D.Bransome, Jr.), Grune and Stratton, Inc., NY (1970).
 

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