Jenkins tetramer production 04-23-10 tetramer... · Jenkins Lab 3 4/25/2010 5) Run digested product...

Jenkins Lab 4/25/2010 1

Jenkins Lab MHC Class II Tetramer Production Protocol – 4/25/10 check for updates at www.jenkinslab.umn.edu

This protocol describes in great pain and detail how to produce soluble I-Ab tetramers containing a covalently linked peptide antigen. The design of the tetramer incorporates Fos-Jun leucine zipper motifs to force dimerize the coexpressed I-Ab α and β chains (Teyton, et. al., J. Exp. Med. 183:2087), and the E.coli BirA signal sequence (Schatz, et. al., Protein Science 8:921) on the α chain to allow for site-specific biotinylation and subsequent tetramerization with fluorochrome-labeled streptavidin. Recently, we have incorporated a "lock-down" strategy of stabilizing the peptide in the MHC binding pocket by introducing a cysteine residue into the linker sequence, which forms a disulfide bridge with another cysteine introduced into a neighboring position in the α1 helix of the MHC alpha chain (J. Kappler, personal communication). Note that this strategy is only feasible if cysteine is not a residue within the antigen peptide sequence.

need revised or additional cartoon


I. Clone Expression Plasmids The covalently-linked peptide antigen is encoded by an N-terminal fusion on the MHCII beta chain. This sequence is flanked on the 5' side by a unique XmaI restriction site (within the leader peptide sequence) and on the 3' side by a unique SpeI restriction site (within the linker sequence). To clone a new peptide sequence, design a set of overlapping oligos encoding the new peptide sequence plus flanking sequences encompassing these restriction sites. For constructs not utilizing the disulfide lock-down strategy, simply substitute the cysteine (C) residue in the linker with another glycine (G).

1) Design overlapping oligos for the insert.

The 5' end of the sense oligo should start 3 bases 5' of the XmaI site and extend 3' for about 70-80 bases. The 5' end of the antisense oligo should start 3 bases 5' of the AvrII site and extend 3' for about 70-80 bases. Request PAGE-purification of these oligos when ordering (requires 100 nmol scale production).

2) Anneal oligos slowly, preferably in a thermocycler. a) Make 100 µM stocks of each oligo in TLE (10 mM Tris pH8, 0.1 mM EDTA) or dH2O. b) Mix together 5 µl of each oligo in a 0.2 ml PCR tube. c) Run the following program in a thermocycler:

i) 95C 30" ii) step -0.1C/s to 4C iii) 4C forever

d) Keep on ice. 3) Run double-stranded product on 2% agarose gel and purify band of expected size using DNA

gel extraction kit (Qiagen 28704). http://www1.qiagen.com/literature/handbooks/PDF/DNACleanupAndConcentration/QQ_Spin/1021422_HBQQSpin_072002WW.pdf.

4) Digest parent vector and new insert fragment with XmaI and SpeI restriction enzymes (New England Biolabs R0180S, R0174S). http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/default.asp

5’-AGCCCCGGGACTGAGGGCCAAGCTGTCCATGCAGCACATGCAGAAATCAATGGCTGTGGAGGTACTAGTGGC-3'!

XmaI SpeI

OVA-2 peptide sequence

sense oligo

antisense oligo

substitute new peptide sequence here

5’-AGCCCCGGGACTGAGGGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGCTGTGGAGGTACTAGTGGC-3’!

3’-TCGGGGCCCTGACTCCCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCGACACCTCCATGATCACCG-5’!

original sequence (pRMHa3 I-Ab beta OVA-2C)

S P G T E G Q A V H A A H A E I N G C G G T S G!

3’-TCGGGGCCCTGACTCCCGGTTCGACAGGTACGTCGTGTACGTCTTTAGTTACCGACACCTCCATGATCACCG-5’!signal peptide linker

substitute new peptide sequence here

-2 -1 1 2 3 4 5 6 7 8 9 +1!MHCII binding pocket position


5) Run digested product on 2% agarose gel and purify band of expected size as in step 4. 6) Ligate purified digested vector and insert fragments (Invitrogen 15224).

http://www.invitrogen.com/content/sfs/manuals/t4dnaligase_1U_man.pdf 7) Transform XLI Blue, DH5α, or equivalent competent cells and plate out on LB agar

containing 100 µg/ml ampicillin (Stratagene 200249). http://stratagene.com/manuals/200249.pdf

8) Miniprep 4-8 colonies (Qiagen 27104). Spot backup colonies on new ampicillin plate for future maxipreps. http://www1.qiagen.com/literature/handbooks/PDF/PlasmidDNAPurification/PLS_QP_Miniprep/1043788_HB_QIAprep_122006.pdf

9) Screen minipreps by sequencing the insert region. Use "I-Ab β peptide" primer (5'-ATCATCTCAGTGCAACTAAA-3'). http://www.bmgc.umn.edu/bmgc/facilities/home.html

10) Maxiprep clone with correct sequence (Qiagen 12262). http://www1.qiagen.com/literature/handbooks/PDF/PlasmidDNAPurification/PLS_QIAfilter/1034638_HB_QIAfilter_112005.pdf


II. Transfect Drosophila S2 Cells Expression of MHC in Drosophila S2 cells is carried out in most part according to the DES Drosphila Expression System from Invitrogen (K5130-01). http://www.invitrogen.com/content/sfs/manuals/des_man.pdf Optional- In vivo biotinylation of MHC is achieved by co-transfection of an expression plasmid for the E. coli BirA enzyme (see Yang, et. al., Human Immunology 65:692-699). 1) Using healthy S2 cells in log growth phase, set up 3 x 106 cells in 3 ml of complete medium

(see recipes) in 60 mm tissue culture dishes. Use medium from Invitrogen (11720-034), as we have had suboptimal growth with media from other vendors. Incubate overnight at 28C. You can place three 60 mm dishes inside a 150 mm dish to provide an additional sterile barrier.

2) Transfect cells using the Calcium Phosphate Transfection Kit (Invitrogen K2780-01) included in the DES kit. A detailed protocol is provided in the DES product manual. http://www.invitrogen.com/content/sfs/manuals/des_man.pdf a) For each transfection, set up and mix in 1.5 ml microfuge tube:

sterile dH2O 236 µl CaCl2 (2M) 240 mM 36 µl pRMHa-3 I-Ab alpha-C BirA plasmid (1 µg/µl) 9 µg 9 µl pRMHa-3 I-Ab beta (peptide)-C plasmid (1 µg/µl) 9 µg 9 µl p18 BirA plasmid (1 µg/µl) (optional) 9 µg 9 µl pCoBlast plasmid (1 µg/µl) 1 µg 1 µl 300 µl

b) Slowly add contents dropwise over the course of 1 min to another 1.5 ml ufuge tube containing 300 µl 2xHEPES HBS while vortexing. This process produces the fine precipitate that mediates transfection of DNA.

c) Incubate mixtures at room temperature for 30-40 min. d) Slowly drop mixture into cell culture while swirling dish. e) Incubate 1 d at 28C. f) Replace medium with fresh complete medium.

i) Pipet medium from the dish into a 15 ml centrifuge tube. ii) Carefully rinse cells with 1 ml fresh medium; add/pool to 15 ml tube. iii) Add 1 ml fresh medium to dish so the cells don't dry out. iv) Spin down tube. v) Resuspend pellet with 2 ml fresh medium; transfer to dish.

g) Incubate 2 d at 28C. 3) Put cells into selection.

a) Repeat procedure described in step II.2.f, but use fresh complete medium containing 25 µg/ml blasticidin (Invivogen ant-bl-5b)(not Invitrogen). Add the blasticidin to the medium first before adding to cells.

b) Incubate 3 days at 28C. c) Gently resuspend the cells by pipetting and transfer 1.5 ml into 13.5 ml fresh complete

medium + blasticidin (25 µg/ml) in a T75 flask (1:10 split). d) Incubate 5 days at 28C. e) Split 1:10 again into a new T75 flask.


f) Incubate 6 days at 28C. 4) At this point, the cells will have been in blasticidin selection for 2 weeks. They should be

ready to scale-up for protein production or sorting. However, from now on they should always be kept in medium containing blasticidin, as they will lose expression over time without selective pressure. Cultures can be maintained indefinitely by splitting them 1:10 in complete medium + blasticidin about once a week. Subcloning S2 cells is very difficult because they do not grow well at low densities.

5) Optional: Sort for highest expressing cells. (see next section) 6) Scale up culture for freezing and protein expression.

a) Split cells 1:10 into two fresh T75 flasks, one containing 15 ml complete medium + blasticidin, another with 15 ml serum free medium (SFM)(see recipes) + blasticidin.

b) Incubate 4-5 days at 28C. c) When the complete medium culture has reached 5-10 x 106 cells/ml (usually about 4-5

days), spin down the cells and resuspend at 107 cells/ml in 45% fresh complete medium, 45% conditioned medium (medium from the culture), and 10% DMSO. Freeze down at least 5 x 1 ml aliquots.


III. [OPTIONAL] Sort for Cells with Highest Levels of Expression (cells must be transfected with BirA plasmid)

1. Set up a 1:10 split of cells in a T75 flask containing 15 ml of complete medium + blasticidin

(25 µg/ml). Set up a flask of parental S2 cells (no blasticidin) in parallel to use as a negative control. In addition, maintain a 5 ml culture of parental S2 cells in a T25 flask throughout this protocol, splitting back 1:10 every 3-4 days, to provide a source of fresh conditioned medium.

2. Incubate 4-5 days at 28C. 3. Add 800 µM CuSO4 (12 µl of 1M solution)(see recipes) and 2 µg/ml D-biotin (30 µl of 1

mg/ml solution)(see recipes). 4. Incubate 4 days at 28C. 5. Perform all steps under sterile conditions. For each sample, pre-warm 15 ml of SFM + 800 µM CuSO4 in 28C incubator.

6. Spin down cells in a 15 ml centrifuge tube. 7. Resuspend pellet in 5 ml cold PBS and then top off to 15 ml. Spin down. 8. Resuspend pellet in 200 µl cold PBS. Add 2 µl of Concanavalin A-biotin (ConA-bio) and

vortex. To make ConA-bio, use the Biotin-XX Microscale Protein Labeling Kit (Invitrogen-Molecular Probes B30010) to biotinylate 100 µg of Concanavalin A (Sigma C7275). The final concentration of biotinylated protein should be approximately 1 mg/ml.

9. Incubate 15 min on ice. 10. Wash with 10 ml cold PBS. 11. Resuspend pellet in 200 µl cold PBS. Add 2 µl of streptavidin(100 µM) for a final

concentration of 1 µM and vortex. 12. Incubate 15 min on ice. 13. Wash with 10 ml cold PBS. 14. Resuspend pellet with 10 ml of pre-warmed SFM + 800 µM CuSO4. Transfer (split equally)

5 ml each to two 100 mm ultra low attachment culture dishes (Corning 3262). Add another 2.5 ml media to each plate for a total of 7.5 ml per plate.

15. Incubate 2-3 h at 28C. 16. Spin down cells in 15 ml centrifuge tube and resuspend with 200 µl cold PBS + 2% FBS.

Add 2 µl of PE-conjugated anti-mouse MHC class II (clone M5/114)(eBioscience 12-5321-81) and vortex.

17. Incubate 30 min on ice. 18. Wash with 10 ml cold PBS + 2% FBS. 19. Resuspend with 2 ml cold SFM (no blasticidin, CuSO4, or D-biotin). Filter through a 5 ml

FACS tube with cell-strainer cap (BD Falcon 352235). Transfer filtered cells to a 5 ml polypropylene FACS tube (BD Falcon 2063). ConA-coated cells will stick to regular polystyrene FACS tubes.

20. Sort for highest pMHC-expressing cells based on PE fluorescence intensity. Collect sorted cells into a 5 ml polypropylene FACS tube containing 0.5 ml cold complete medium + blasticidin. Use the S2 parental cells as a negative control to set the sorting gate. Generally, the obviously positive cells will constitute about 0.5 - 5 % of the total population. Collect as many positive cells as possible, with no fewer than 10,000.


21. Spin down the sorted cells. Very carefully aspirate the supernatant down to about 200 µl. Resuspend with 900 µl complete medium + blasticidin (25 µg/ml) + fungizone (250 ng/ml)(Invitrogen 15290-018) and transfer to one well of a 24-well tissue culture plate. Rinse the tube with 900 µl of fresh conditioned medium from parental S2 cells and transfer to the same well. Note that the final concentration of blasticidin will be about half of the normal dose. Add 2 ml of sterile PBS to the surrounding wells to minimize evaporation of culture medium.

22. Incubate at 28C. Monitor the cells carefully for growth and contamination daily. Every third day, carefully pipet out about 1.5 ml of the medium without disturbing the cells and replace with 750 µl fresh complete medium + blasticidin + fungizone and 750 µl fresh conditioned medium from S2 parental cells.

23. When the cells grow to a point where they're covering about half the surface area of the well, transfer the entire culture to a T25 flask containing 3.5 ml fresh complete medium + blasticidin + fungizone (final volume ~5 ml). You do not need to use conditioned medium anymore.

24. Incubate at 28C. When the cells reach a density of 5 - 10 x 106 /ml, proceed back to step II.6. for the scale-up for freezing and protein expression.


IV. Scale Up Cell Cultures / Induce Expression

1) When the SFM culture reaches 5-10 x 106 cells/ml, split 1:10 into two T225 flasks each containing 50 ml SFM + 1% pen/strep + 20 µg/ml gentamycin + blasticidin (25 µg/ml).

2) Incubate until cultures reach 5-10 x 106 cells/ml, usually about 5 days at 28C. 3) Split cultures 1:10 into a 3 L spinner flask or 2 L shaker flask containing 1.0 L SFM +

pen/strep + gentamycin + blasticidin. 4) Incubate at 28C with constant spinning or shaking at 125 rpm. Lower speeds (~100 rpm) are

okay too, but the cell densities will be slightly lower. 5) When the culture reaches 1.0-1.5 x 107 cells/ml (usually about 5 days), induce expression by

adding 800 µM CuSO4 (800 µl of 1M solution). For cells transfected with the BirA expression plasmid for in vivo biotinylation, also add 2 µg/ml D-biotin (2 ml of 1 mg/ml solution) (Pierce 29129).

6) Incubate 10-14 days at 28C, 125 rpm.


V. Purify Soluble pMHC Soluble I-Ab heterodimers are purified from cell culture supernatant via nickel affinity chromatography targeting the 6x His epitope on the C-terminus of the I-Ab beta chain. Eluted pMHC is then further purified from contaminants via size exclusion chromatography (Sephacryl S-300). A. Isolate cell culture supernatant. 1) Spin down induced S2 cells from step IV.6. (500 ml conical tubes at 2500 rpm for 10 min in

a Beckman tabletop centrifuge). Spin down supernatant again in new tubes. 2) Filter supernatant first through 3 µm pore-size Whatman paper (Whatman 1003-240), and

then directly through a 0.45 µm vacuum filter. Aggregated material in the supernatant can make this a slow process. Multiple filters may be needed.

3) Add 0.1% azide and store at 4C. Sometimes aggregates will form in the filtered supernatant as it is stored in the cold. If this happens, re-filter it.

B. Perform nickel affinity chromatography. Nickel affinity chromatography is carried out using the Novagen His-Bind Purification Kit (Novagen-EMD Biosciences 70239-3). http://www.emdbiosciences.com/docs/docs/PROT/TB054.pdf It is important to use a chelating resin based on iminodiacetic acid (IDA) instead of nitriloacetic acid (NTA) reactive groups. This is because free copper ions in the cell culture supernatant will efficiently compete with His-tagged protein/ion complexes for binding to NTA groups, but not to IDA groups (see Lehr, et. al., Protein Expr Purif 19:362-368). 1) Add 4 ml of well mixed His-Bind nickel resin slurry to a 1 x 10 cm glass column (Kontes

420400-1010) clamped on a ringstand. The resin is supplied as a 50% suspension slurry, so 2 ml of packed resin will remain in the column after the liquid portion has drained away. This amount of resin should have a binding capacity of about 16 mg protein (8 mg per ml of resin).

2) Wash the column by adding 6 ml dH2O and letting drain. 3) Add about 1/4 inch layer of 425-600 µm diameter glass beads (Sigma G9268) to the top of

the resin bed to protect the top surface from disruption as fluids are added. 4) Charge the column by adding 10 ml of 1X Charge buffer and letting drain. The color of the

resin should turn from white to blue. 5) Wash the column by adding 6 ml of 1X Bind buffer and letting drain 6) Affix a closed stopcock to the bottom of the column. Add a few ml of culture supernatant to

the top of the resin, at first by slowly running it down the inside of the column to prevent disruption of the resin. Secure the cap on top of the column.

7) Place the bottle containing the culture supernatant on a shelf a few feet above the top of the column. Set up a length of small diameter flexible tubing connecting the bottle of culture supernatant to the cap on top of the column. We recommend using 1/16 in (inner diameter) tubing (Tygon ALC00002) with a short (~2 in) length of 1/8 in (inner diameter) tubing (Tygon ALC00007) at the end to act as a coupling for attachment to the nipple on the end of the column cap. Make sure that the lowest point in the tubing loop hangs below the bottom of the column. This will prevent the column from running dry after the bottle has emptied.


8) To prime the tubing, remove the tubing from the nipple on the column cap. Using a syringe, draw fluid from the bottle to near the end of the tubing, then clamp the tubing about 2 inches from the end with a hemostat. Remove the syringe, then re-attach the end of the tubing to the nipple on the column cap.

9) Unclamp the top tubing and open the stopcock. The fluid should start dripping from the bottom of the column. Use the stopcock to adjust the flow rate to about 2 ml per minute. Let the column drip overnight, collecting the flow-through fraction in a new bottle. The safety loop should prevent the resin from drying out once the supernatant bottle has emptied. Leaving the resin in a dry state is bad.

10) Once the supernatant has passed over the column, close the stopcock, clamp the top tubing and remove the cap. Transfer the column on the ringstand to a bench in the lab. Open the stopcock to let any remaining supernatant drain from the resin.

11) Wash the column with 5 ml 1X Bind buffer (10 mM imidazole). 12) Arrange six 13 x 100 mm glass tubes in a row on a rack. Position the rack containing the

elution tubes such that the first tube is under the column. Add 12 ml of 1X Elute buffer (1M imidazole) to the top of the column. Collect 2 ml elution fractions into each tube. In other words, collect 2 ml, then manually move the rack to the 2nd tube, collect 2 ml, etc. until all 12 ml of buffer has passed through the column and you have 2 ml of fluid in 6 tubes. Close the stopcock.

13) Regenerate the column by adding 6 ml 1X Strip buffer and letting drain. The resin should change in color from blue to white. Close the stopcock and add another 2 ml to the top of the column. Replace the cap on the column, and put the column back in the cold room for storage.

14) Assess the pMHC content in each fraction by Western blot (2 µl per lane). Pool the fractions containing the most pMHC, usually the first 3-4. If you want to save time, you can skip this step and just harvest all of the elution.

15) Concentrate the pooled sample to ~200 µl by spinning through an Amicon Ultra-15 50 kD concentrating filter (Millipore) at ~1500g (3500 rpm Beckman tabletop centrifuge).

C. Perform size exclusion chromatography Size exclusion chromatography is performed using a 1 x 50 cm glass column containing about 60 ml of packed Sephacryl S-300 resin (GE Amersham Pharmacia). 1) Open the clamp on the tubing at the bottom of the column, place the tubing in a beaker, and

let about 50 ml of PBS + 0.1% azide (PBSA) drip through from the reservoir placed on a shelf about 2 feet above the column top. Clamp the tubing at the bottom of the column with a hemostat to stop the flow.

2) Use a hemostat to clamp the tubing attached to the top of the column, then unscrew the lid from the top of the column and remove ALL of PBS sitting above the top of the column bed using a 10 ml pipette without sucking up any glass beads.

3) Attach the tubing from the bottom of the column (which should be clamped shut with a hemostat) to the port on the fraction collector. Put 30, 13x100 mm glass tubes in the fraction collector and use the Manual Advance to position the first tube under the port. Hit Start twice to reset the fraction collector to tube 1. Set the device to collect 25 drops (~1.5 ml) per tube.

4) Unclamp the tubing at the bottom of the column. Fluid will now slowly drip through the column. The glass beads will change in appearance as the fluid moves past them and they dry


out. Just as the glass beads go dry, add the sample drop wise in a volume of 0.2-0.5 ml to the center of the glass bead stack.

5) Let the sample run into the column until the glass bead stack goes dry again. 6) Add 10 ml of PBSA to the top of the column by slowly running it down the side of the

column TAKING CARE NOT TO DISTURB THE BEADS. 7) Screw the lid back on and unclamp the hemostat from the tubing on the top of the column. 8) Collect 30 fractions at a rate of about 1 drop per 7 sec. It will take about an hour total. Run

about 50 ml of PBSA into a beaker, then clamp the tubing at the bottom of the column to stop it.

9) Set the spectrophotometer to 280 nm with the UV light on. Blank on the lst tube, then read the OD on all 30. Two peaks should be apparent, one centered on fraction 13-15 containing high molecular weight S2 proteins, another at 18-20 containing pMHC.

10) Pool the fractions corresponding to the pMHC (second) peak and concentrate to about 0.5 ml

by spinning through an Amicon Ultra-4 50 kD concentrating filter (Millipore). 11) Measure the OD280 (try a 1:20 dilution first) and calculate the pMHC concentration. The

extinction coefficient for pMHC is ~1.0 cm-1(mg/ml)-1 (i.e. an OD280 of 1.0 corresponds to a concentration of 1.0 mg/ml). The molecular weight of pMHC is about 70 kD.

concentration = OD280 x dilution factor / 1.0 (final units in mg/ml) (divide by MW for units in molarity)

S2 cell impurities

pMHCII (alpha-beta dimers)

undimerized beta chain

harvest


VI. Assess pMHC biotinylation Because the sample still contains a significant amount of impurities and the biotinylation process is not 100% efficient, your concentration based on the OD280 reading will be an overestimate of the true concentration of biotinylated pMHC. The following titration assay will empirically determine how much of your pMHC sample should be mixed with SA-PE to create fully-armed tetramers. Set up the following reactions in microfuge tubes: Tube Total amount of

pMHC per tube Volume of pMHC to be added to each tube

Total amount of SA-PE per tube

Volume of SA-PE to be added to each tube

1 1 µg 5 µl of 200 µg/ml None 0 2 1 µg 5 µl of 200 µg/ml 5 pmol 5 µl of 1 µM 3 1 µg 5 µl of 200 µg/ml 2.5 pmol 5 µl of 500 nM 4 1 µg 5 µl of 200 µg/ml 1.25 pmol 5 µl of 250 nM 5 1 µg 5 µl of 200 µg/ml 0.62 pmol 5 µl of 125 nM 6 1 µg 5 µl of 200 µg/ml 0.31 pmol 5 µl of 62.5 nM 7 None 0 5 pmol 5 µl of 1 µM 1) Prepare 35 µl of pMHC at 200 µg/ml, based on the OD280 measurement from step V.C.12. 2) Prepare a two-fold dilution series of streptavidin-phycoerythrin (SA-PE, Prozyme PJRS25 or

PJRS27) starting with 30 µl of 1 µM. For example, if this reagent is supplied at 7.2 µM SA, mix 4.2 µl of this with 25.8 µl PBSA. Then mix 10 µl of this stock with 10 µl of PBSA to make a 20 µl stock of 500 nM. Repeat to make a 1:2 serial dilution series extending down to 62.5 nM.

3) Add 5 µl of the 200 µg/ml pMHC to each tube, then 5 µl of the SA-PE dilutions as shown in the above table. Incubate for 30 minutes to an hour at room temperature.

4) Add 4 µl of 4X native PAGE sample buffer (see recipes) to each tube, then run on a 4-15% Tris-glycine acrylamide gel using native PAGE running buffer (see recipes). Use 10 µl of Precision Plus Kaliedoscope molecular weight markers (BioRad 161-0375) in the outer lanes. Run at 150V about 1 h.

5) Transfer the proteins to nitrocellulose. 6) Western blot with a 1:10,000 prep of SA-AF680 (Invitrogen S21378) as a probe. 7) Use the Odyssey Infrared Imager to detect SA-AF680 binding. Lane #1 should give a single

strong band at about 80 kD, corresponding to biotinylated pMHC (this band runs a little higher than normal on a native gel). No band should be present in Lane #2 because all of the biotinylated pMHC should be bound to SA-PE. The 80 kD band will eventually re-appear as the SA-PE concentration falls and can no longer occupy all of biotinylated pMHC. The SA-PE amount where the biotinylated pMHC band just re-appears defines the saturation point. If no saturation point is observed, then repeat the assay making according adjustments to the amount of pMHC loaded in each lane. Be conservative with your assumptions so that you always err on the side of underestimating the true concentration of biotinylated pMHC.


8) To calculate the concentration of biotinylated pMHC in your sample, first multiply the molar

amount of SA-PE in the saturation point by a factor of 4 to determine the molar amount of biotinylated pMHC that was loaded in that lane. For example, in the above blot, the saturation point was Lane #3, which contained 2.5 pmol of SA-PE and 10 pmol of biotinylated pMHC. Divide this molar amount of biotinylated pMHC by the 5 µl volume in which it was delivered to determine the concentration of biotinylated pMHC in the "200 µg/ml" diluted stock of pMHC monomer prepared in step 1. In this example, this would be 10 pmol ÷ 5 µl = 2 µM biotinylated pMHC. Finally, multiply this concentration by the dilution factor used to generate the "200 µg/ml" stock to calculate the concentration of biotinylated pMHC in the original sample.

Note that this concentration is only as accurate as the listed concentration of SA-PE from the manufacturer. We have noticed disparities between titration results obtained with the same pMHC stock and different streptavidin products from different manufacturers. While we trust the reported values from Prozyme enough that we use the same titration results for different production lots of streptavidin as well as different types of streptavidin (e.g. SA-APC), ideally it is best to titrate your sample to the particular tube of streptavidin that you will be using to tetramerize your pMHC.

250 150

100 75

50

37

1 2 3 4 5 6 7

pMHC-bio


VII. Tetramerize pMHC 1) The goal at this point is to mix 4 moles of biotin-labeled pMHC with 1 mole of SA-PE to

generate a fully multimerized tetramer. The procedure in the previous section will define this point. For example, if in the preceding example, the biotinylated pMHC band just re-appears in Tube 3, then you would saturate all the biotin-binding sites on 2.5 pmol of SA-PE with 1 µg of pMHC. Therefore, to make tetramer, you would want a pMHC:SA-PE ratio of at least 6:1.

2) 100 µl of SA-PE makes a reasonable amount of tetramer. For a typical 8 µM stock of SA-PE, this corresponds to 800 pmol. Therefore, in the above example, add at least 6 x 800 = 4.8 nmol of your original pMHC stock to saturate all the biotin binding sites on the SA-PE. To be conservative, you may want to add about 20% more than the empirically derived saturating amount of pMHC to ensure the generation of only fully-armed tetramers.

3) Incubate at room temperature for 30 minutes. 4) Purify the tetramer from unincorporated pMHC by running the sample through the S-300

size-exclusion column as described in step V.C.

5) Two peaks should be apparent, one centered on fraction 12-14 containing PE-labeled

tetramer, and another at 17-19 containing both biotinylated and unbiotinylated pMHC. 6) Pool the fractions corresponding to the tetramer (first) peak and concentrate to about 0.25 ml

by spinning through an Amicon Ultra-4 100 kD concentrating filter (Millipore). 7) Measure the OD566 (try a 1:10 dilution first) and calculate the PE concentration. The

extinction coefficient for PE is 1.96 cm-1µM-1 (i.e. an OD566 of 1.96 corresponds to a concentration of 1.0 µM).

concentration = OD566 x dilution factor / 1.96 (final units in µM)

tetramer

free pMHCII

harvest


8) Ideally, there should be exactly one molecule of SA conjugated to each molecule of PE in the SA-PE stock. However, this stoichiometry is usually slightly off. The data sheet for each lot of SA-PE should specify the individual concentrations of SA and PE. Convert the measured PE concentration of your sample to that of SA by multiplying by the [SA]:[PE] ratio obtained from the data sheet. Because SA constitutes the core of the tetramer, the concentration of SA should be the actual concentration of the tetramer.

9) Optional: Dilute the concentration of tetramer to 1.0 µM, and store away multiple aliquots at 4C. This is a convenient stock concentration from which to work with during experiments.

10) The final staining concentration for cells is usually about 10 nM, but every production lot of tetramer should be titrated in a pulldown experiment on mice immunized with the corresponding antigen (usually peptide + LPS i.v. for 8 days).


Recipes Complete S2 Medium: Schneider's Drosophila Medium 500 ml (Invitrogen 11720) Fetal Bovine Serum 10 % 50 ml (heat-inactivated @ 56C 20 min) Pen/Strep 100 U/ml each 5 ml of 100X Gentamycin 20 µg/ml 1 ml of 10 mg/ml Serum Free Medium (SFM): Express Five Serum Free Medium 1 L (Invitrogen 10486-025) Pen/Strep 100 U/ml each 10 ml of 100X Gentamycin 20 µg/ml 2 ml of 10 mg/ml 1M CuSO4: CuSO4 (copper (II) sulfate) pentahydrate 1 M 12.5 g (Sigma C8027) dH2O qs 50 ml pass through sterile 0.2 µm filter 1 mg/ml D-biotin: D-biotin 1 mg/ml 50 mg (Pierce 29129) dH2O 49.8 ml 1N NaOH ~190 µl to pH 7 pass through sterile 0.2 µm filter 4x Native Lam's Sample Buffer: Tris HCl pH 6.8 200 mM 5 ml of 1 M Glycerol 60 % 15 ml Bromophenol Blue 20 µg/ml 500 µg dH2O qs 25 ml 5X Native Tris-Glycine SDS-PAGE Running Buffer: Tris-base 125 mM 15.1 g Glycine 1.25 M 94 g dH2O qs 1.0 L

Jenkins tetramer production 04-23-10 tetramer... · Jenkins Lab 3 4/25/2010 5) Run digested product...

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