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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN DETECTION: GEL ELECTROPHORESIS

TRICHLOROACETIC ACID (TCA) PRECIPITATION OF RADIOLABELED PROTEIN AND QUANTIFICATION BY SCINTILLATION SPECTROSCOPY

Trichloroacetic Acid (TCA) Precipitation of Radiolabeled Protein and Quantification by Scintillation Spectroscopy
 
Overview
A method for the quantification of radiolabeled protein, based on the ability of Trichloroacetic Acid (TCA) to precipitate protein onto Whatman glass fiber filters. Filters can then be subjected to scintillation spectroscopy to quantitate radiolabeled proteins. Scintillation spectroscopy is a complex multi-step process that begins when weak beta or gamma radiation is emitted from a source. The particle slows down and stops in the scintillator (scintillation cocktail), leaving a trail of excited atomic or molecular species along its track. Some of these excited species return to their ground state in a process that involves the emission of a photon of light. The photon energy (or the wavelength of the light) is distributed over an emission spectrum that is characteristic of the particular scintillation material. Some fraction of the light leaves the scintillator through an exit window where it is measure by a light sensor. The remaining surfaces of the scintillator are provided with a reflective coating so that the light that is originally directed away from the exit window has a high probability of being reflected from the surfaces and collected. The net result of this sequence of steps, each with its own inefficiency, is the creation of a relatively limited number of charge carriers in the light sensor which are reported as counts per minute (CPM). CPM is related to disintegrations per minute (DPM) by the counting efficiency. Counting efficiency can be calculated using a radioactive standard sample, calculating the DPM of the standard and measuring the CPM of the standard. Counting efficiency is equal to CPM divided by DPM and is reported as a percentage (i.e. a 22% counting efficiency in a sample with a CPM of 12,000 would indicate a DPM of 100,000).
 
Procedure
A. Sample and Filter Apparatus Preparation

1. Aliquot approximately 5 to 10 μl of radiolabeled sample onto Whatman 2.4 cm GF/C glass fiber filters.

2. Place the filters sample side up on card board paper covered with aluminum foil and attach the filter to the card board sheet using a push pin.

3. Adjust the height of the filter so that it does not touch the surface of the aluminum foil.

4. Allow filters to completely air dry (preferably under a heat lamp).

B. TCA Washing of Filters

1. Rinse the filter apparatus with H2O and allow to dry

1. Assemble the filter apparatus (see Hint #1).

2. Rinse the filter well(s) with a few ml of the 5% TCA Solution.

3. Using forceps, place a filter (prepared in Section A) in the filter manifold well with the sample facing up (see Hint #2).

4. Add approximately 5 ml of 5% TCA Solution and allow the liquid to run through the filter with no vacuum applied for a couple of seconds.

5. Apply a gentle vacuum to the manifold and allow the solution to completely run through the filter.

6. Repeat Steps #B4 and #B5 two more times.

7. Apply a full vacuum until the filters are dry.

8. Repeat Steps #B4 and #B5 using 95% Ethanol.

9. Apply a full vacuum until the filters are dry.

10. Using forceps remove the filters from the manifold and attach to the card board covered by aluminum as before (see Step #A2 and #A3).

11. Allow filters to dry for approximately 45 min (preferably using a heat lamp).

C. Scintillation Counting of Filters

1. Place filter samples in a scintillation vial.

2. Add 5 ml of Scintillation Cocktail and gently mix (see Hint #3).

3. Make use of a scintillation radioisotope to calculate the counting efficiency (refer to the Overview section of this protocol for a description of counting efficiency).

4. Set the counting time and windows for the radionucleotide (see Hint #4).

Solutions
95% (v/v) Ethanol   Keep on ice
5% TCA Solution   Keep on ice
0.75% (w/v) Sodium Pyrophosphate (Na4P2O7), see Hint #5
5% (v/v) Trichloroacetic acid
 
BioReagents and Chemicals
Ethanol
Sodium Pyrophosphate
Trichloroacetic Acid
Toluene
Scintillation Cocktail
 
Protocol Hints
1. A variety of vacuum manifolds are available ranging from a single filter well to a multi-filter well system. Assemble the filter apparatus according to the manufacture's instructions. A filtration system has four major parts: 1) a manifold, 2) a reservoir, 3) a vacuum trap and 4) a vacuum source. The manifold consists of the filter well and a base upon which the filter rests. A solution is added to the filter well where it is driven through the filter by the vacuum source and into the reservoir. Usually a vacuum trap is included in a filtration system. The trap (usually an Erhlenmyer flask) is in between the vacuum source and the reservoir; therefore if the reservoir fills to capacity the solution is "trapped" before it reaches the vacuum source. Never allow a liquid to enter the vacuum source since this will severely damage the vacuum pump.

2. If the filter apparatus has multiple wells, make sure to block the follow of air through wells that do not contain a filter sample. If the filter well has a valve, turn it to the off position. If the filter well does not have a valve, stop the vacuum flow by placing a rubber stopper in the well.

3. It is not necessary to use scintillation fluid to count 32P-labeled samples. The Cerenkov radiation emitted from such samples can be detected by a scintillation counter set to monitor the tritium (3H) window. Although the absolute number of counts is not the same between the two methods (Cerenkov counting is less than half as efficient), they will be in proportion from sample to sample. When reporting the lack of scintillation in the counting process, mention that radioactivity was determined as Cerenkov radiation in a scintillation counter.

4. The counting time will need to be determined empirically since the specific activity of the protein will vary depending upon the quantity and specific activity of the radiolabel. The window positions will vary depending on whether you are quantifying 35S or 32P. See the manufacturer's manual for the window setting for your specific isotope.

5. It is not necessary to add 0.75% (w/v) Na4P2O7 when working with 35S labeled protein.