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Lotte Bjergbæk (ed.), DNA Repair Protocols, Methods in Molecular Biology, vol. 920,DOI 10.1007/978-1-61779-998-3_38, © Springer Science+Business Media New York 2012

Chapter 38

Analysis of Inhibition of DNA Replication in Irradiated Cells Using the SV40 Based In Vitro Assay of DNA Replication

George Iliakis , Emil Mladenov , Ya Wang , and Hong Yan Wang

Abstract

The deleterious effects of DNA damage on DNA replication have been demonstrated in many model systems and the mechanisms of the resulting inhibition have been a research focus for at least 40 years. Moreover, recent studies have identi fi ed several major components of the S-phase checkpoint, providing a mechanistic background for understanding the basis of inhibition of the initiation and elongation steps of DNA replica-tion after DNA damage. Yet several aspects of the underlying biochemical mechanisms remain unresolved including the characterization of the enzymatic activities involved in checkpoint activation and the coordina-tion of this process with DNA repair. Helpful for the delineation of the mechanism of the S-phase check-point is the observation that factors inhibiting DNA replication in vivo can be found in active form in extracts prepared from irradiated cells, when these are tested using the simian virus 40 (SV40) assay for in vitro DNA replication. In this assay, replication of plasmid DNA carrying the minimal origin of SV40 DNA replication is achieved in vitro using cytoplasmic cell extracts and SV40 large tumor antigen (TAg) as the only noncellular protein. Here, we describe protocols developed to measure in vitro DNA replication with the purpose of analyzing its regulation after exposure to DNA damage. The procedures include the preparation of components of the in vitro DNA replication reaction including cytoplasmic extracts from cells that have sustained DNA damage. The assay provides a powerful tool for investigating the effect of distinct agents acting either by inducing lesions in the DNA, or by inhibiting the functions of checkpoint proteins. Nevertheless, the fact that several steps of DNA replication initiation are carried out in this in vitro assay by TAg and not the corresponding cellular factors, might be considered as a limitation of the approach.

Key words: DNA replication , S-phase Checkpoint , SV40 , T-antigen , In vitro assay , Cell extract

Inhibition of DNA replication in eukaryotic cells is one of the earliest effects of radiation to be reported and quanti fi ed. Elucidation of the mechanism causing this inhibition has been a research focus in several laboratories for at least four decades. A signi fi cant develop-ment for these efforts was the recognition that the mechanism of inhibition has a direct ( cis -acting) and an indirect ( trans -acting)

1. Introduction

592 G. Iliakis et al.

component (for reviews, see ref. 1, 2 ) . Whereas the direct component is thought to derive from radiation-induced DNA damage that alters chromatin structure and inhibits DNA replication in cis , the indirect component is attributed to the activation by DNA damage of regulatory processes that inhibit DNA replication in trans . The latter is equivalent to the activation of a checkpoint during the S-phase ( 2– 7 ) .

The realization that a checkpoint is activated in S-phase after induction of DNA damage has led to intensive studies aiming at its genetic and biochemical characterization. The best documented genetic alteration that affects the regulation of DNA replication in response to radiation exposure is found in individuals with the hereditary genetic disorder ataxia telagiectasia (AT). AT cells fail to inhibit DNA replication in response to radiation suggesting that the underlying gene, ATM, a key member of the PIKK family of protein kinases, is involved in the regulation of DNA replication (for reviews, see ref. 8, 9 ) . Other experiments implicate ATR, another member of PIKK family in the regulation of DNA replica-tion following the generation of other forms of DNA damage. Recent studies have identi fi ed several additional components of this checkpoint response and there is strong interest in its bio-chemical characterization.

Promising for the delineation of the mechanism of the S-phase checkpoint is the observation that factors that inhibit DNA replica-tion in vivo can be found in active form in extracts prepared from irradiated cells, when these are tested for replication activity using the simian virus 40 (SV40) assay for in vitro DNA replication ( 10– 12 ) . In this assay, replication of plasmids carrying the minimal origin of SV40 DNA replication is achieved in vitro using cytoplasmic cell extracts and SV40 large tumor antigen (TAg) as the only noncellular protein ( 13– 16 ) . It is thought that cellular proteins function in this assay in the same manner as in vivo. A further advantage of the SV40 assay is its ability to initiate DNA replication at a de fi ned origin, a function that cannot be achieved in other in vitro system, where only the elongation step of DNA replication can typically be studied.

The assay has been extremely successful in the fi eld of DNA replication and has led to the characterization of a number of factors involved in eukaryotic DNA replication. In a similar way, the assay could also help in the biochemical characterization of important components of the regulatory pathway activated in response to DNA damage (e.g., ( 17, 18 ) ). While the assay is limited by the fact that a single noncellular protein, TAg, carries out functions assigned to different families of proteins in eukaryotic cells ( 19 ) , its poten-tial for the biochemical characterization of checkpoint responses has not been exhausted and modi fi cations mitigating some limita-tions may be possible.

Here, we describe protocols developed to measure in vitro DNA replication with the purpose of analyzing its regulation after

59338 Analysis of Inhibition of DNA Replication in Irradiated Cells…

exposure to DNA damage. The required procedures include: (1) Preparation of cytoplasmic extract from cells that have sus-tained DNA damage; (2) Preparation of the SV40 large TAg; (3) Preparation of supercoiled plasmid DNA carrying SV40 origin of DNA replication; (4) Assembly of in vitro replication reactions; (5) Assay of DNA replication using incorporation of radioactive pre-cursors, and of the DNA replication products using gel electropho-resis. Graphic outlines of the important steps of the procedures are summarized in Fig. 1 .

1. Minimum Essential Medium (MEM) modi fi ed for suspension cultures (S-MEM), supplemented with 5 % fetal bovine serum (FBS) or iron-supplemented bovine serum and antibiotics (penicillin 100 U/mL, streptomycin 100 μ g/mL).

2. Hypotonic buffer solution: 10 mM HEPES-KOH, pH 7.5 (stock 0.6 M, pH 7.5 at room temperature (RT)), 1.5 mM MgCl 2 (stock 0.5 M), 5 mM KCl (stock 3 M). Immediately before use add 0.2 mM PMSF (stock 100 mM in ethanol), 0.5 mM dithiothreitol (DTT) (stock 1 M in H 2 O; store at −20 °C), and 20 mM β -glycerophosphate.

3. High salt buffer: 100 mM HEPES-KOH, pH 7.5, 1.4 M KCl, and 15 mM MgCl 2 .

2. Materials

2.1. Preparation of HeLa-Cell Extract

Fig. 1. Outline of the individual steps and preparations required for assembling DNA repli-cation reactions using the SV40 system for in vitro DNA replication and for evaluating their outcome.

594 G. Iliakis et al.

4. Dialysis buffer: 25 mM Tris–HCl, pH 7.5 at 4 °C (stock 1 M, pH 7.5 at 4 °C), 10 % glycerol, 50 mM NaCl (stock 5 M), 1 mM EDTA (stock 0.5 M, pH 8.0). Immediately before use add 0.2 mM PMSF, 0.5 mM DTT, and 20 mM β -glycerophosphate.

5. 10× Phosphate-buffered saline (PBS): 14.7 mM KH 2 PO 4 , 42.9 mM Na 2 HPO 4 , 1.37 M NaCl, 26.8 mM KCl. (pH of 10× PBS is 6.8).

6. Trypsin solution: 1× PBS pH 7.4 (pH 1× PBS is 7.4), 10 mM EDTA pH 8.0.

7. Microcarrier spinner fl asks of 30 L nominal volume (e.g., Bellco Glass Inc.).

8. Microcarrier magnetic stirrers (e.g., Bellco Glass Inc.). 9. Tissue culture dishes (100 mm). 10. Dounce homogenizer with B pestle, 50 mL.

1. Hybridoma cell line PAb419; clone L19 generated by Harlow et al. should be used ( 20 ) . It produces a monoclonal antibody that recognizes the amino terminal region of TAg and is used for the preparation of immunoaf fi nity columns employed in the puri fi cation of TAg. The cell line can be requested from Dr. Harlow, and is used to produce antibody that can be puri fi ed using standard procedures ( 21 ) .

2. Insect cells (Sf9). Available from ATCC. 3. Baculovirus 941T, Autographa californica expressing TAg.

The 941T virus was constructed by Lanford et al. using a TAg cDNA ( 22 ) and can be requested from the author. The proce-dures used to prepare stocks of the virus and to measure its infectivity in pfu/mL are described in specialized protocols and the reader is referred to these publications for more infor-mation, e.g., ( 23 ) .

4. TD buffer: 25 mM Tris–HCl pH 7.4, (stock 1 M, pH 7.4), 136 mM NaCl (stock: 5 M), 5.7 mM KCl (stock 3 M), 0.7 mM Na 2 HPO 4 (stock 0.2 M).

5. Buffer B: 50 mM Tris–HCl, (stock 1 M, pH 8.0), 150 mM NaCl (stock 5 M), 1 mM EDTA (stock 0.5 M, pH 8.0), 10 % glycerol, 1 mM PMSF (stock 0.1 M), 1 mM DTT (stock 1 M).

6. Buffer C: 50 mM Tris–HCl pH 8.0, (stock 1 M, pH 8.0), 500 mM LiCl (stock 1 M), 1 mM EDTA (stock 0.5 M, pH 8.0), 10 % glycerol, 1 mM PMSF (stock 0.1 M), 1 mM DTT (stock 1 M).

7. Buffer D: 10 mM PIPES-NaOH, pH 7.4, (stock 1 M, pH 7.4, dissolved in 1 M NaOH), 5 mM NaCl (stock 5 M), 1 mM

2.2. Preparation of TAg

59538 Analysis of Inhibition of DNA Replication in Irradiated Cells…

EDTA (stock 0.5 M, pH 8.0), 10 % glycerol, 1 mM PMSF (stock 0.1 M), 1 mM DTT (stock 1 M).

8. Buffer E: 20 mM triethylamine, 10 % glycerol, pH 10.8. Prepare just before use.

9. Buffer F: 10 mM PIPES-NaOH, (stock 1 M, pH 7.0), 5 mM NaCl (stock 5 M), 0.1 mM EDTA (stock 0.5 M, pH 8.0), 10 % glycerol, 1 mM PMSF (stock 0.1 M), 1 mM DTT (stock 1 M).

10. Reagents: Sodium borate, dimethylpipelimidate, ethanolamine, triethylamine, merthiolate, NP-40.

11. Chromatography supplies: Protein A agarose, Sepharose 4B-Cl (Invitrogen, GE Healthcare), syringes (5 mL), or EconoColumns with ID of 0.75 cm (Bio-Rad).

12. Supplies and equipment for SDS-PAGE. 13. Two 1 L and one 250 mL microcarrier spinner fl asks (Bellco). 14. Material for protein determination using the Bradford assay (a

ready to use solution is available from Bio-Rad).

Several plasmids that carry the minimum origin of SV40 DNA rep-lication are available and can be used for this purpose, e.g., pSV01 Δ EP ( 24 ) ; pSV010 ( 25 ) ; and pJLO ( 26 ) . Large quantities of these plasmids can be prepared using cesium chloride ethidium bromide gradients. The description of these methods are beyond the scope of the present protocol and can be found in publications describing molecular biology protocols ( 23, 27 ) .

1. Replication reaction solution (5×): 200 mM HEPES-KOH, pH 7.5, 40 mM MgCl 2 , 2.5 mM DTT, 200 mM Phosphocreatine, 15 mM ATP, 1 mM CTP, 1 mM GTP, 1 mM UTP, 0.5 mM dATP, 0.125 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP. Prepare 10 mL replication reaction solution and freeze in small aliquots.

2. Creatine phosphokinase stock: 2.5 mg/mL prepared in 50 % v/v glycerol. Phosphokinase together with phosphocreatine present in the replication reaction solution form an ATP regen-eration system that is required for DNA replication.

3. Salmon sperm DNA. 4. Reagents: EDTA, trichloroacetic acid (TCA), sodium dodecyl

sulfate (SDS). 5. Enzymes: Proteinase K, RNase A. 6. Scintillation counter. 7. Gel electrophoresis equipment. 8. Glass fi ber fi lters (GF/C, Whatman).

2.3. Preparation of Supercoiled Plasmid DNA Carrying the SV40 Origin of DNA Replication

2.4. Assembly of In Vitro Replication Reactions and Product Analysis

596 G. Iliakis et al.

The method described here is a modi fi cation of a method originally developed by Dignam et al. ( 28 ) , and allows the preparation of cell extract from 10 L of cell suspension. Higher or lower amounts of extract can be prepared by appropriate scaling. Ionizing radiation or radiomimetic chemicals can be used for the generation of dam-age in the DNA (see Note 1). We obtained satisfactory results using a 3 h treatment with 0.5 μ g/mL camptothecin, an effective inhibi-tor of type I topoisomerases. We also use routinely 10–40 Gy of 25 MV X-rays from a linear accelerator. The details for the latter treatment depend heavily upon the type of equipment used. It is therefore impractical to describe them here. Investigators with access to such equipment are advised to request the assistance of radiation physicists for dosimetry and other details on the irradia-tion protocol. Standard X-ray-producing equipment (50–250 kV) cannot be used to irradiate volumes of the magnitude described here due to their low penetration characteristics. Whatever the solu-tion for the irradiation problem, it is important to keep in mind that disturbance in the cell culture has to be kept to a minimum before and after irradiation for reproducible results. Signi fi cant reductions in temperature, changes of the growth vessels, centrifu-gations, etc., should be avoided. Extracts can be processed for repair at a speci fi c time after irradiation. The precise timing will depend upon the type of experiments and its speci fi c goals. We prepare extracts 0–3 h after the treatment to induce DNA damage.

1. Grow HeLa cells at 37 °C for 3 days in twenty- fi ve 100 mm tissue culture dishes prepared at an initial density of 3 × 10 6 cells/dish in 20 mL S-MEM supplemented with serum and antibiotics. The fi nal density after 3 days of growth should be ~20 × 10 6 cells/dish, giving a total of 5 × 10 8 cells in 25 dishes (see Note 2).

2. During that step the cells are adherent and are attached to the growing vessel, therefore the trypsinization step is required. Detach the cells from all dishes using 0.05 % Trypsin in Trypsin solution and resuspend in 10 L of pre-warmed complete growth medium in a 30 L nominal volume microcarrier fl ask, thoroughly pre-gassed with 5 % CO 2 in air. The initial cell con-centration should be ~5 × 10 4 cells/mL. Place in a warm room at 37 °C and provide adequate stirring (~40–60 rpm).

3. Allow cells to grow for 4 days, to a fi nal concentration of 4–6 × 10 5 cells/mL. Do not exceed this concentration.

4. Collect cells by centrifugation (8 min at 2,500 × g ). Collection should be fast and is best done using a refrigerated centrifuge that can accept 1 L bottles (e.g., Beckman J6-MI). All further processing should be carried out at 0–4 °C.

3. Methods

3.1. Preparation of Cell Extract

59738 Analysis of Inhibition of DNA Replication in Irradiated Cells…

5. Rinse twice in PBS and centrifuge (5 min at 500 × g ). Determine the packed cell volume (pcv) (~12–15 mL total).

6. Resuspend cell pellet in 5 pcv volumes of hypotonic buffer solution and centrifuge quickly (5 min, 500 × g ). Cells swell and the pcv approximately doubles.

7. Determine the new pcv. Resuspend cell pellet in 3 pcv of hypo-tonic buffer and disrupt in a Dounce homogenizer (20 strokes, pestle B). It is advisable to test cell disruption using a phase contrast microscope.

8. Add 0.11 volumes of high salt buffer and centrifuge 3,000 × g for 20 min.

9. Carefully remove supernatant and centrifuge at 100,000 × g for 1 h.

10. Place the resulting extract (S100) in dialysis tubing with an MW cutoff of 10–14 kDa and dialyze overnight against 50–100 volumes of dialysis buffer.

11. Collect extract. Centrifuge at 15,000 × g for 20 min to remove precipitated protein (see Note 1). Aliquot and snap freeze using liquid nitrogen. Immediately store at −70 °C and keep a small aliquot for determining protein concentration using the Bradford protein assay (Protein assay, Bio-Rad).

Good quality TAg is essential for ef fi cient replication in vitro of plasmids carrying the SV40 origin of DNA replication. Investigators can either obtain this protein from commercially available sources, or can prepare it in the laboratory using available reagents. The method of preparation described here is essentially the one described by Simanis and Lane ( 29 ) , and can conveniently be sepa-rated in the preparation of the TAg immunoaf fi nity column and the puri fi cation of TAg from extracts of sf9 cells infected with the baculovirus 941T which expresses TAg.

1. Mix 5 mg PAb419 antibody with 2 mL wet protein A beads. Incubate at room temperature for 1 h with gentle rocking.

2. Wash the beads twice with 20 mL 0.2 M sodium borate (pH 9.0) by centrifugation at 1,000 × g for 5 min.

3. Resuspend the beads in 20 mL 0.2 M sodium borate (pH 9.0), mix and remove 100 μ L bead-suspension for assaying coupling ef fi ciency. Add solid dimethylpipelimidate to bring the fi nal concentration to 20 mM.

4. Mix for 30 min at room temperature on a rocker and remove 100 μ L suspension of the coupled beads.

5. Stop the coupling reaction by washing the beads with 20 mL 0.2 M ethanolamine (pH 8.0) and incubate for 2 h at room temperature in 0.2 M ethanolamine with gentle rocking.

3.2. Preparation of TAg

3.2.1. Preparation of TAg Immunoaf fi nity Column

598 G. Iliakis et al.

6. Spin down and resuspend the beads in PBS. Add 0.01 % merthiolate if extensive storage is anticipated. At this point, beads are ready for use for the puri fi cation of TAg (if the quality control performed next is positive).

7. Check the ef fi ciency of coupling by boiling in Laemmli sample buffer the samples of beads taken before and after coupling. Run the equivalent of 1 and 9 μ L beads from both samples on a 10 % SDS-PAGE gel and stain with Coomassie Brilliant Blue G250. Due to the coupling procedure TAg is covalently attached to the Protein A agarose beads. Good coupling is indicated by bands indicating the mobility of IgG heavy chain (55 kDa) in the samples obtained before but not in the samples obtained after coupling procedure.

8. Prepare immunoaf fi nity column by pouring beads into a 5 mL syringe, or a 0.75 cm in diameter EconoColumn.

1. Grow enough sf9 cells to prepare 1 L of cell suspension at 2 × 10 5 cells/mL. Distribute the cell suspension in two 1 L microcarrier spinner fl asks (500 mL cell suspension per spin-ner) and incubate at 27 °C under gentle spinning (1–1.5 revo-lutions per second). Allow cells to grow until they reach a concentration of 2 × 10 6 cells/mL (3–4 days) (see Note 3).

2. Spin cells for 5 min at 500 × g and carefully return supernatant to spinner fl asks.

3. Resuspend cells in enough volume of virus stock to reach a multiplicity of infection equal to 10 pfu per cell. Place cell sus-pension in a 250 mL spinner fl ask and allow attachment of virus to the cells by gentle stirring at 27 °C for 2 h.

4. Return cell suspension to the original spinner fl asks and incu-bate for 48 h at 27 °C under gentle stirring to allow for protein expression. We have noted that good protein expression is also achieved by adding the viral stock directly into the cell culture and incubating for 48 h.

5. Harvest cells by centrifugation at 500 × g for 5 min and resus-pend in 25 mL TD buffer.

6. Centrifuge at 500 × g for 5 min and resuspend in 30 mL buffer B. Add 10 % NP-40 to a fi nal concentration of 0.5 %.

7. Place on ice for 30 min. Invert tube several times every 10 min.

8. Centrifuge for 10 min at 18,000 × g in a corex tube. At this stage, cellular extract can be removed and quickly frozen at −70 °C for use at a later time for puri fi cation of TAg. When needed, thaw the extract quickly by immersing in 4 °C water bath. Do not allow extract to warm-up above 4 °C. All subse-quent steps should be carried out in a cold room.

3.2.2. Puri fi cation of TAg from Extracts of sf9 Cells

59938 Analysis of Inhibition of DNA Replication in Irradiated Cells…

9. Save a 50 μ L aliquot of the cell extract and load the remaining material onto a 2 mL Sepharose 4B-Cl column equilibrated with buffer B. Elution can be achieved either by gravity, or with the help of a peristaltic pump giving a fl ow rate of approx-imately 1 mL/min. This step retains proteins binding nonspeci fi cally to sepharose media.

10. Allow the fl ow through of step 9 to pass through a 2 mL pro-tein A agarose column equilibrated with buffer B. This can be achieved either by gravity, or with the help of a peristaltic pump giving a fl ow rate of approximately 1 mL/min. This step retains material binding nonspeci fi cally to protein A.

11. Allow the fl ow through of step 10 to pass through a 2 mL immunoaf fi nity column of protein A agarose coupled with antibody PAb419. This can be achieved either by gravity, or with the help of a peristaltic pump at a fl ow rate of approxi-mately 0.5 mL/min. This step retains TAg from the cell extract. Optionally, to increase the binding ef fi ciency the loop may be designed to allow cellular extract containing TAg to pass through the column multiple times.

12. Repeat step 11. Save fl ow through. 13. Wash immunoaf fi nity column with 200 mL of buffer C. This is

best achieved with a peristaltic pump giving a fl ow rate of approximately 2 mL/min.

14. Wash immunoaf fi nity column with 100 mL of buffer D. This is best achieved with a peristaltic pump giving a fl ow rate of approximately 2 mL/min.

15. Elute with buffer E. Collect 0.5 mL fractions in tubes contain-ing 25 μ L of 1.0 M PIPES-NaOH pH 7.0. Place fractions on ice (see Note 4).

16. Wash immunoaf fi nity column with 20 mL buffer E, and then with 40 mL buffer B. Column can be reused four to fi ve times.

17. Measure protein concentration using the Bradford protein assay.

18. Combine fractions containing protein. Dialyze overnight against 2 L buffer F.

19. Test quality of puri fi cation by SDS-PAGE followed by silver staining. Test activity for in vitro SV40 DNA replication. The procedure yields 1–2 mg TAg per liter of sf9 cell culture.

Standard procedures can be used for the preparation of supercoiled plasmid DNA. It is preferable to purify the DNA using a two step puri fi cation procedure on CsCl 2 /ethidium bromide gradient. Detailed protocols for this purpose are beyond the scope of the pres-ent protocol and can be found other sources of protocols ( 23, 27 ) .

3.3. Preparation of Supercoiled Plasmid DNA Carrying the SV40 Origin of DNA Replication

600 G. Iliakis et al.

1. Assemble 50 μ L reactions by mixing in an Eppendorf tube kept on ice 5 μ L reaction buffer, 2.5 μ L creatine phosphate, 200–400 μ g extract protein, 1 μ g TAg, 0.3 μ g superhelical plasmid DNA, 0.001 μ Ci/mL ( α - 32 P)dCTP; adjust volume to 50 μ L with nuclease-free H 2 O. Extracts from untreated and treated cells should be used in parallel so that the results obtained can be directly compared.

2. Incubate reactions at 37 °C for 1 h. Longer or shorter incuba-tions can also be used if information on the kinetics of replica-tion is desired.

3. Terminate reactions by adding EDTA to a fi nal concentration of 20 mM.

4. Add 25 μ g of denatured salmon sperm DNA and mix well. 5. Add 1 mL of cold 10 % TCA to precipitate nucleoprotein com-

plexes. Mix well. 6. Collect precipitate onto Whatman® GF/C glass fi ber fi lters.

Wash three times with 10 mL cold 10 % TCA. Wash four times with 10 mL deionized water.

7. Add 5 mL scintillation fl uid. Measure incorporated activity in a scintillation counter.

1. To analyze the DNA replication products by gel electrophore-sis, add to the stopped replication reactions 0.1 % SDS.

2. Digest with RNase A (20 μ g/mL) for 15 min at 37 °C. 3. Add proteinase K (200 μ g/mL) and incubate at 37 °C for

30 min. 4. Purify DNA either by extraction in phenol/chloroform fol-

lowed by precipitation in ethanol, or by using commercially available DNA puri fi cation systems.

5. Separate in 1 % agarose at 6.5 V/cm for 2 h. Electrophoretic conditions may need to be modi fi ed depending on the plasmid size used in the assay. For optimal resolution, reduce fi eld intensity to 1 V/cm.

1. The effect of the DNA damage inducing agent on DNA repli-cation in vitro can be variable. We found that this is usually due to the growth conditions (overgrown cultures), or to the absence of phosphatase inhibitors in the prepared extract. We routinely add β -glycerophosphate since we found it to signi fi cantly improve the reproducibility. Other phosphatase

3.4. Assembly of In Vitro Replication Reactions and Evaluation of Replication Activity

3.5. Analysis of the Replication Products

4. Notes

60138 Analysis of Inhibition of DNA Replication in Irradiated Cells…

inhibitors, as well as the use of protease inhibitors should be considered if reproducibility problems persist.

2. The preparation of a good extract depends strongly on the quality of the cells used. When cell growth is not optimal, or when cells overgrow, low replication activity may be obtained and the inhibition in extracts of treated cells may be subopti-mal. To ensure optimal growth we routinely take the following measures: (a) Carefully test different batches of serum to fi nd one with good growth characteristics. HeLa cells have a gen-eration time of less than 20 h when grown as a monolayer, and less than 24 h when grown in suspension, under optimal growth conditions. (b) Use cells grown in dishes to start the suspension cultures for extract preparation. This helps to reduce clumping occurring after extensive growth in suspen-sion. (c) We follow cell growth daily, and collect cells for extract preparation when they reach a concentration of 4–6 × 10 5 cells/mL. (d) We measure cell cycle distribution by fl ow cytometry. A high percentage of S-phase cells (~25 % for HeLa cells), sug-gests that the cell culture is still in an active state of growth.

3. Optimal cell growth is also a prerequisite of a successful prepa-ration of TAg. We fi nd that sf9 cells grow more consistently if kept in suspension. Transfer from a monolayer state to a sus-pension state is usually associated with a shock that takes the cells some time to overcome.

4. We have observed that 20 mM triethylamine may not elute all bound TAg from the immunoaf fi nity column. If this proves to be the case, increase in triethylamine concentration (up to 100 mM), or alternative eluting methods (see ref. 21 ) should be considered. However, it should be kept in mind that such alternatives may reduce TAg activity.

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