an understanding of the techniques

SIGNALLING DOWNSTREAM OF THE EPIDERMAL GROWTH FACTOR

AssignmentTutorOnline

RECEPTOR TYROSINE KINASE

AIMS OF THE PRACTICAL

At the end of the practical you should have an understanding of the techniques involved in the design, construction and expression of GST fusion proteins in E. coli and their use in the biochemical characterization of protein-protein interactions involved in growth-factor signalling. You should also understand the principles of SDS-PAGE, western blotting and the use of phospho-specific antibodies to study growth-factor signalling pathways.

The specific objectives are:

1. To express a GST fusion of the SH2 domain of PLC inE. coli and purify it by affinity chromatography.

2. To demonstrate the EGF-dependent association between the PLC SH2 domain and the EGF receptor by co-precipitation and western blotting.

3. To determine the time course of activation of the EGF receptor and downstream signalling molecules by EGF, using western blotting with phospho-specific antibodies.

INTRODUCTION

Signalling pathways downstream of the EGF Receptor

Epidermal Growth Factor (EGF) is able to increase cell proliferation in many cell types by binding to one of a large family of receptors that possess intrinsic tyrosine kinase activity. EGF binding to the EGF receptor tyrosine kinase generates multiple intracellular signals that are responsible for increasing the rate of cell division. These include phosphorylation of the receptor itself and many other cellular proteins on tyrosine residues, the release of calcium from intracellular stores and the activation of several protein kinase cascades, most notably the ERK or MAP kinase pathway. EGF binding to the EGF receptor (EGFR) causes receptor oligomerisation, activation of the kinase domain and autophosphorylation of a number of tyrosine residues on the intracellular portion of the receptor, particularly those at the C-terminus. These include tyrosines 845, 992, 1068, 1086, 1148 and 1173. Many of the tyrosines phosphorylated provide docking sites for intracellular signalling proteins that are recruited to the receptor by virtue of their Src Homology 2 (SH2) domains, which bind to specific phosphotyrosine residues. Some of these recruited proteins, for example phospholipase C (PLC), are then phosphorylated by the receptor. Other SH2 domain containing proteins, such as Grb2, are adaptor proteins allowing the activation of other pathways downstream. The recruitment, phosphorylation and activation of PLC by the EGFR is responsible for the mobilization of intracellular calcium, whilst recruitment of Grb2 is required for the activation of the ERK pathway. Both of these signalling pathways play a critical role in the regulation of cell growth by growth factors and must be tightly regulated to prevent uncontrolled cell growth. This is brought about by a series of coordinated and highly specific protein-protein interactions that are regulated by protein phosphorylation (Fig. 1). In this practical we will study the interaction of the EGFR with PLC using GST fusion proteins produced in E. coli. We will also use antibodies that recognize phosphorylated proteins to investigate the time course of pathway activation by EGF.

Fig. 1 – Activation of signalling pathways downstream of the EGFR

GST fusion protein production in E. coli.

To aid their purification, proteins expressed in bacteria may be fused to other proteins that have the ability to bind to small molecules. Glutathione S-transferase (GST), binds specifically to the short peptide, Glutathione (GSH), which can be chemically coupled to sepharose beads. These beads may then be used to purify GST-fusion proteins by affinity chromatography as shown in the diagram below (Fig. 2). Purified proteins may either be eluted by the addition of excess glutathione or the immobilized fusion protein used directly in an experiment.

Fig. 2 – Purification of GST-PLC SH2 by affinity chromatography

To produce GST-fusion proteins in bacteria, the cDNA encoding the protein is cloned into a bacterial expression vector which is then transformed into an appropriate strain of E. coli. In our experiments we shall be using the inducible bacterial expression plasmid, pGEX-3X (Fig. 3).

Fig. 3 – pGEX-3X bacterial expression vector

pGEX-3X contains cloning sites that allow the in-frame insertion of a cDNA sequence to generate a construct that can be used to express an N-terminal GST-fusion protein in E. coli. GST fusion protein expression is under the control of the strong Ptac promoter, which is repressed by the lacl repressor protein, also encoded by the plasmid. Expression of the fusion protein is induced (de-repressed) by the addition of the lactose analogue isopropyl-β-thiogalactoside (IPTG) to the bacterial culture.

In our experiment we will express and purify two proteins in E. coli. GST alone (pGEX with no cDNA insert) and a fusion protein in which an SH2 domain from phospholipase C (PLC) is fused to GST (GST-PLC-SH2).

Co-precipitation of the EGFR with GST fusion proteins

The GST and GST-PLC-SH2 fusion proteins will be used to study the interaction of the SH2 domain of Phospholipase C with the epidermal growth factor receptor. To do this, the GST-fusion proteins will be immobilized on glutathione sepharose beads and incubated with extracts from MDA-MB-468 cells that have been treated with or without EGF. After washing the beads to remove proteins that do not bind, any EGFR that does bind to the fusion proteins (Fig. 4) may then be detected by SDS-PAGE and western blotting for the EGFR.

Fig. 4 – Co-precipitation of the EGFR with a GST-PLC SH2 fusion protein

Western Blotting

Specific proteins may be detected in complex mixtures by western blotting. Proteins are first separated according size by SDS-PAGE and then transferred to a nitrocellulose filter. The filter is then incubated with a specific primary antibody. The primary antibody is then detected by incubation of the blot with a secondary antibody that binds to the primary antibody. The secondary antibody is covalently coupled to the enzyme Horseradish Peroxidase (HRP) which allows visualization of the western blot by incubation with an enhanced chemiluminescent (ECL) substrate which produces light which can be detected and quantified (Fig.5).

Fig. 5 – Western Blotting

Experiment I – Co-precipitation of the EGFR and PLC SH2 domain

The aim of this experiment is to purify a GST-fusion of the PLC SH2 domain expressed in E. coli and use it to demonstrate the EGF dependent interaction of the EGFR with PLC The experiment involves three steps; the expression and purification of GST-fusion proteins in E. coli, the preparation of cell extracts from MDA-MB-468 cells and the co-precipitation experiment itself.

Method

Day 1: Preparation of GST-PLC SH2 fusion protein

1. Using sterile technique, set up two 50 ml Falcon tubes with 10 ml of 2YT medium add Ampicillin (AMP) to a concentration of 50µg/ml. The AMP stock is 50 mg/ml. Label them GST and GST-SH2. Inoculate the tubes with a 2 ml aliquot of the overnight cultures of GST and GST-SH2 respectively. Incubate the cultures for 1 h at 37°C in the shaking incubator.

2. After 1 h remove a 100 l sample of each culture to an 1.5 ml tube labelled “GST – IPTG” or “GST-SH2 – IPTG”. To the remaining culture add IPTG to a final concentration of 1mM. The IPTG stock is 1 M. Incubate for a further 3 h at 37°C. Pellet the cells in the 100l sample by spinning at 13,000 rpm in a benchtop centrifuge for 1 min. Remove the supernatant, discarding it in a beaker of disinfectant, and resuspend the cell pellet in 30 µl H₂0. Add 15 l SDS-PAGE sample buffer (SPSB) and heat in a heating block at 95-100°C for 3 min. Store at -20°C.

During your incubation period pour two SDS-PAGE gels

3. At the end of the 3 h incubation period, remove another 100 l sample and process as before, in tubes labelled “GST + IPTG” and “GST-SH2 + IPTG”. You should now have samples of induced (+ IPTG) and uninduced (– IPTG) cultures for both GST and GST-SH2 fusion proteins. Retain these samples in the -20°C freezer for later use.

4. Pellet the cells in the remaining cultures in the 50ml tubes by centrifugation for 5 min at 3000 rpm.

5. Discard all of the supernatant in disinfectant and resuspend the bacterial pellet completely by adding 1 ml of ice-cold Extraction Buffer (EB), and mixing vigorously by pipetting the solution carefully in and out of a pipette tip multiple times. Transfer to a 1.5ml tube and add Triton X-100 to 1% using the stock solution provided (i.e. stock solution is 100% Triton X-100) and mix thoroughly but gently to avoid froth. Freeze/thaw the bacterial suspension by immersing the tubes in liquid N₂, then thawing, three times to help break the cells open. Leave on ice for 20 min.

6. Remove the insoluble material in the bacterial lysate by centrifugation at 13,000 rpm for 5 min at 4°C in the chilled benchtop centrifuge. Transfer the supernatants to fresh tubes and keep on ice. The pellet can be discarded.

7. You should now have two bacterial lysates containing GST and GST-SH2 which need to be purified by affinity chromatography. To do this resuspend the sepharose thoroughly before pipetting, using a p1000, cut the end off a blue pipette tip to help with pipetting, add 200 l of the slurry of Glutathione Sepharose to each tube of lysate. Put these tubes in a 30ml universal tube labelled with your bench number and the time you want them returned. Give this tube to a demonstrator to tumble on the roller in the cold-room at 4°C for 30 min.

8. Pellet the sepharose beads with a brief 10 sec spin (13,000 rpm) in a benchtop centrifuge and place the tubes on ice. Remove and discard the supernatant carefully without losing sepharose beads.

9. Wash the glutathione sepharose pellet by resuspending it in 1 ml of ice-cold Extraction Buffer (EB) mixing the contents of the tubes thoroughly. Re-pellet the sepharose beads and carefully discard the supernatant. Repeat the washes twice more, resuspending the final pellet in 1 ml of Extraction Buffer (EB).

10. You should now have two tubes, each containing approximately 100 µl (settled volume) of glutathione sepharose with either purified GST or GST-SH2 bound in a final volume of just over 1 ml. Store these tubes at 4°C (fridge) overnight for use in the experiment tomorrow.

11. Work out the volumes of all the components needed to make 35mls of Triton Lysis buffer (recipe in appendix) so that you can be ready to make it tomorrow.

Day2 (am): Preparation of cell extracts for GST pull-down experiment

You will be provided with two dishes of MDA-MB-468 cells, a tumour cell line that over-express the EGFR. These cells have been serum starved overnight to deprive them of growth factors. You will treat these cells with or without EGF and make cell extracts to be used in the co-precipitation experiment.

1. Make up 35 ml of a working solution of Triton Lysis Buffer (TLB) from the 2X stock solution and stocks of additional components as indicated in the Appendix. Chill the buffer on ice before using it.

2. Take one dish of cells from the incubator and pour off the culture medium, inverting the dish onto some paper towel to drain the last of the medium. Wash the cells once by gently adding 10 ml of ice-cold Phosphate-Buffered Saline (PBS). Remove all of the PBS by pouring it off and inverting the dish on some paper towel (this is important).

3. Put 1 ml of ice-cold Triton Lysis Buffer (TLB) onto the plate and scrape cells using the cell scraper provided into the buffer. Gently pipette the cell extract up and down several times to disperse the cells and place the mixture in an 1.5 ml tube labelled “-EGF”, keep it on ice.

3. You are going to treat a second dish of cells with 200 ng/ml EGF (stock is 100 µg/ml) for exactly 5 min at 37°C in the incubator. Do this by taking the EGF on ice to the incubator (don’t bring the cells back to your bench). Clearly label a second dish of cells with your bench number. Add the EGF directly to the 10ml of serum-free medium on the cells and mix gently by swirling the dish.

4. It is important to extract the cells exactly 5 min after adding EGF, to extract them quickly and to keep the cell extract cold. Therefore, after the 5 min incubation with EGF, quickly pour off the culture medium, inverting the dish onto some paper towel to drain the last of the medium. Wash the cells carefully with 10 ml of ice-cold Phosphate-Buffered Saline (PBS). Remove all of the PBS by inverting the dish on some paper towel (this is important).

5. Put 1 ml of ice-cold Triton Lysis Buffer (TLB) onto the plate and scrape cells into the buffer. Gently pipette the cell extract up and down several times to disperse the cells and place the mixture in a 1.5 ml tube labelled “+EGF” on ice. Incubate the extracts on ice for 5 min.

6. You should now have two cell extracts, one from control (-EGF) and one from EGF treated cells (+EGF). Spin the 1.5 ml tubes in a benchtop centrifuge at full speed for 5 min at 4°C to pellet the cell debris. Using a pipette, and being careful not to disturb the cell debris in the pellet at the bottom of the tube, transfer the supernatants to fresh, pre-labelled tubes and store on ice for the next part of the experiment. Pellets can be discarded.

Co-precipitation of the EGFR and PLC SH2 domain

For this you will need the cell extracts from control (-EGF) and EGF treated (+EGF) cells that you have just prepared and the purified GST fusion proteins (GST and GST-SH2) coupled to glutathione sepharose beads that you prepared yesterday and stored at 4°C.

1. Take 40 l samples from both the control and EGF cell extracts and add 20l of SDS-PAGE sample buffer (SPSB). Heat at 95-100°C for 3 mins. Store these samples at -20°C.
2. Taking your tubes from yesterday containing the GST and GST-SH2 bound to thesepharose beads, resuspend the beads by briefly vortexing. Using a pipette tip with the end cut off to aid pipetting, immediately, before the beads have time to settle, and as accurately as you can, divide the mixture into two by pipetting 500 µl into a fresh tube and leaving the remaining 500 µl in the original tube. You now have 4 tubes, 2 for GST and 2 for GST-SH2.
3. Spin the 4 tubes of beads briefly to pellet them. Check by eye that there is an equal amount of beads in each of the tubes. Remove the supernatant and resuspend beads in 1 ml TLB. Wash them once more with 1 ml of TLB, pellet the beads, remove the last supernatant completely and add 150 µl of TLB. Check again that there is still an equal amount of beads in each of the 4 tubes.
4. Using these tubes containing your beads label as in the top row of this table. Add your clarified cell extracts as indicated. Label the tubes clearly and keep them on ice.

Tube label	GST -EGF	GST+EGF	GST-SH2 -EGF	GST-SH2 +EGF
beads GST / GST-SH2	GST (200l)	GST (200l)	GST-SH2 (200l)	GST-SH2 (200l)
Cell Extract -EGF / +EGF	-EGF (400l)	+EGF (400l)	-EGF (400l)	+EGF (400l)
Total Volume	600l	600l	600l	600l

5. Mix the contents of the 4 tubes thoroughly put into a 50ml tube and label with your bench number and time you want them returned. Give to a demonstrator to incubate with tumbling for at least 2 h on the roller in the cold room at 4°C.
6. After two hours, pellet the beads in the tubes with a short 10 second spin in the benchtop centrifuge.
7. Remove the supernatant with a pipette, taking care not to disturb the sepharose beads. This contains the GST fusion protein and any co-precipitated EGFR.
8. Resuspend the beads in 1 ml of ice-cold TLB. Keep the tubes on ice when they are not in the centrifuge. Repeat this wash procedure twice more with ice-cold TLB, being careful not to lose any of the sepharose beads.
9. Remove the final supernatant without allowing the sepharose pellet to dry out and add 30 l of SDS-PAGE sample buffer (SPSB) and 20 l of distilled water. Mix the tubes vigorously then heat at 95-100°C for 3 mins. Store these samples in the -20°C freezer.
10. Experiment II – Day2 (pm): Pathway analysis by western blotting with phospho-specific antibodies

In this experiment you will determine the time course of activation of signalling events downstream of EGF binding to its receptor, by western blotting with antibodies that detect phosphorylated proteins. MDA-MB-468 cells that over-express the EGFR will be treated with EGF and extracts prepared using buffers that preserve the phosphorylation state of cellular proteins. Proteins in the extracts will be separated by SDS-PAGE, transferred to nitrocellulose membranes and probed with antibodies specific for phospho-tyrosine, phospho-ERK, phospho-PLC and tubulin. Each group will be using only one of these antibodies.

Method

Preparation of Cell Extracts

You have been provided with a 6-well tissue culture plate containing some serum starved MDA-MB-468 cells in 2 ml of serum-free medium and a stock solution of EGF (100 g/ml).

Make sure you still have at least 5 ml of the Triton Lysis Buffer (TLB) that you made earlier.

1. Treat the cells for 0, 1, 5, 10, 20 and 40 min with 200 ng/ml EGF by adding the appropriate amount of EGF directly to the medium and mixing by gently swirling the plate. Incubate the plate of cells at 37°C. Arrange it so that all of the cells can be extracted at the same time, i.e. stagger the addition of EGF to each well. Do not add EGF to 0 min well.
2. At the end of the incubation, remove the medium from the cells and wash each well quickly with 2 ml of ice-cold Phosphate-Buffered Saline (PBS). Remove all of the PBS, place the dish on ice, and extract the cells quickly by scraping them into 50 µl of ice-cold Triton Lysis Buffer (TLB). Gently pipette the cell extract up and down several times to disperse the cells and place each extract in a separate, labelled, 1.5 ml tube on ice. It is important to extract the cells exactly at the right time after adding EGF, to extract them quickly and to keep the cell extract cold.
3. Incubate the tubes on ice for 5 min. Pellet the cell debris by spinning the extract at 14,000 rpm for 5 min in a benchtop centrifuge at 4°C. Add 40 l of the clarified extract (supernatant) to 20 µl SDS-PAGE sample buffer (SPSB). Heat at 95-100°C for 3 mins. Store these samples in the freezer.

Day3: SDS-PAGE and western blotting

Experiment I

1. Collect together all of the SDS-PAGE samples; the glutathione sepharose pellets, the cell extracts from Day2, and the bacterial pellets (± IPTG) from Day1 prior to SDS-PAGE gel electrophoresis.
2. Run the experiment I samples, along with some pre-stained molecular weight standards on a 9% SDS-PAGE gel at 180V, constant voltage, until the blue tracking dye just reaches the bottom of the gel (approximately 1 hour).
3. Once the run is complete, cut the gel in half between the 55 and 72 kDa pre-stained Molecular Weight Markers (see diagram in Appendix). If you are unsure about this, consult a demonstrator.
4. The lower half of the gel, which should include the GST fusion proteins, should be stained for 30 min in Coomassie Blue solution in a tray on a rocking platform. Make sure you are wearing gloves when handling the gel and stain. Unpolymerised acrylamide in the gel and methanol in the stain are both toxic.
5. Carefully pour off stain and rinse gel once with tap water. Destain the gel in Destain solution I (10% acetic acid, 40% methanol). At the end of the day tip off destain and replace with fresh destain solution II (10% acetic acid). Leave overnight until the protein bands can be seen clearly. Store your gel in distilled water until you are ready to photograph it.
6. The upper half of the gel will be western blotted for the EGFR. Using the method in the Appendix, transfer the upper half of the gel, which should include any bound EGFR, onto blotting membranes, block the membrane and incubate overnight in anti-EGFR antibody.

Experiment II (gel to be run after experiment 1 gel)

1. Collect together your experiment II samples prior to SDS-PAGE gel electrophoresis.
2. Run the samples, along with some pre-stained molecular weight standards on a 9% SDS-PAGE gel at 180V, constant voltage, until the blue tracking dye just reaches the bottom of the gel (approximately 45 min).
3. Using the method in the Appendix, transfer the whole gel onto nitrocellulose membrane, block the membrane and incubate overnight in primary antibody (anti-phosphotyrosine, anti-phospho-ERK, anti-phospho-PLC or anti-tubulin. You will be told which one to make up and use).

Day4: Completion of Western Blotting

Develop the blots as described in the western blotting section of the Appendix. They may need to be incubated with different secondary antibodies depending on the primary antibody used. Depending on imager availability some blots will be imaged on Day5.

APPENDIX

1. Cell Extraction

Triton Lysis Buffer (TLB)

To make up a working solution TLB is provided as a 2X stock which needs additional components adding and making up to its final volume with distilled water. Chill the buffer on ice.

The final concentrations should be:

1X TLB (20 mM Hepes, pH 7.5 / 137 mM NaCl / 25 mM β-glycerolphosphate / 2 mM Sodium Pyrophosphate / 2 mM EDTA / 10% glycerol): These are all contained in the 2X stock at twice the concentration needed.

The additional components you will need to add are:

1% (v/v) Triton X-100 (stock solution is 100%)

2 g/ml of PAL – Pepstatin, Antipain, Leupeptin (2 mg/ml stock of all three in 50% EtOH)

2 mM Benzamidine (1M stock in H₂O)

0.5 mM Dithiothreitol (1M stock in H₂O)

1 mM Sodium Orthovanadate (Na₃VO₄) (100 mM Stock in H₂O)

1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) (200 mM stock in H₂O)

Finally work out how much additional water needs adding to make your final volume.

Epidermal Growth Factor (EGF)

Stock solution is 100 g/ml in PBS

2. SDS-PAGE Gel Electrophoresis

You will be shown how to assemble the SDS-PAGE apparatus and the solutions you will need to make up are listed below. N.B. Unpolymerised acrylamide is a neurotoxin and you must wear gloves when handling it. You should pour two 9% SDS-PAGE gels; one for today’s experiment and one for tomorrow. You will also need to make up 1 L of SDS-PAGE Running Buffer.

N.B. Unpolymerised acrylamide is a neurotoxin. Wear gloves and eye protection. Add the components in the order listed. The mixture will not start to polymerise until the APS is added, so add this last just before you want to pour the gel mix between the glass plates. Remember that you will need enough of the mixture to make two gels.

With the help of a demonstrator, load the samples and some Molecular Weight Markers onto the gel (10 µl of the markers per lane should be sufficient). Run the gel at 180V, constant voltage, until the blue tracking dye just reaches the bottom of the gel (approximately 45 min).

9% Separating gel mix

For each gel
H₂O 4.35 ml
1.5 M Tris/pH 8.8 2.6 ml
30% Acrylamide/0.8% Bis 3 ml
TEMED 5 l
10% Ammonium Persulphate (APS) 50 l

Water-saturated Butanol – Overlay the separating gel with 500l of water –saturated butanol after pouring. Remove this before pouring the stacking gel.

Stacking gel mix

For each gel
H₂O 3 ml
0.5 M Tris/pH 6.8 1.25 ml
30% Acrylamide/0.8% Bis 0.67 ml
TEMED 5 l
10% Ammonium Persulphate (APS) 50 l

SDS-PAGE Running Buffer

3g Tris
14.4g Glycine
1g SDS
Make up to 1 litre with distilled water

SDS-PAGE Molecular Weight Markers

(Fermentas PageRuler Prestained Protein Ladder #SM0672)

3. Western Blotting

To transfer proteins from your gel onto the blotting membrane you will need about 100 ml of some freshly diluted Western Transfer Buffer (WTB), 2 sheets of filter paper and a piece of blotting membrane cut to the approximate size of the gel. You should wear gloves when handling the blotting membrane to avoid leaving fingerprints.

1. Set up the semi-dry blotting apparatus as shown by a demonstrator. Transfer at 10V (~75 mA per gel) for 1 h.
2. After transfer is complete mark the position of each of the bands of the molecular weight markers with a pencil. Incubate for 1 h in 15 ml of the appropriate Blocking Buffer (see primary antibody table below)in the plastic tray provided on a rocking platform. Note that the blocking buffer is the same buffer that the primary antibody is diluted in.
3. Discard the Blocking Buffer, but don’t let the membrane dry out. Incubate overnight at 4°C in 15 ml of diluted Primary Antibody.
4. After the overnight incubation, pour off the primary antibody and wash the membrane three times, each with approximately 100 ml Phosphate-Buffered Saline/0.1%Tween-20 (PBS-T), over a period of about 30 min (3 x 10 min).
5. Remove the PBS-T and incubate the blot for 1 h with 15 ml of the appropriate Secondary Antibody. Check which secondary to use with your primary antibody.
6. Remove the secondary antibody and wash again with 3 x 100 ml PBS-T. Keep in PBS until imager is available.
7. Just before imaging, incubate the membrane in the chemiluminescent substrate (this is prepared for you by mixing equal volumes of Luminol and peroxide solution) for 5 minutes. Drain the membrane of excess liquid, then take an image on the imaging system.

Western Transfer Buffer (WTB) – prepared for you

Dilute from 10X stock
Add MeOH to 10% on dilution to 1X (add MeOH last)

PBS-T

Diluted from a 10X PBS stock. You will need about 1l for each blot.
PBS/0.1% (v/v) Tween-20

Blocking Buffer

PBS/0.1% (v/v) Tween-20/3% BSA or 5% milk (same as primary antibody)

Primary Antibodies

Rabbit anti-EGFR @ 1:1000 in PBS-T_0.1/3% BSA
Mouse anti-PY99 (anti-phospho-tyrosine) @ 1:750 in PBS-T_0.1/3% BSA
Mouse anti-phospho-ERK @ 1:1000 in PBS-T_0.1 5% milk
Rabbit anti-phospho-PLCγ (Y783) @ 1:1000 in PBS-T_0.1/3% BSA
Mouse anti--tubulin @ 1:5000 in PBS-T_0.1/5% milk

Secondary Antibodies

Anti-mouse IgG AP conjugate @ 1:5,000 in PBS-T_0.1/3% BSA
Anti-rabbit IgG AP conjugate @ 1:5,000 in PBS-T_0.1/3% BSA