IL-2 ELISPOT

Materials Required
·  Peptides of desired specificity (stock at 10mM in DMSO - may be aliquotted and stored at -20ºC)
·  Phytohemagglutinin (PHA; optional) for use as a control for cytokine production (Sigma #L8902)
·  Purified anti-IL-2 capture antibody (e.g. clone IL2-I, Mabtech #3440-3)
·  Biotin-conjugated anti-IL-2 detection antibody (e.g. clone IL2-II, Mabtech #3440-6)
·  Streptavidin-alkaline phosphatase 1mg/ml (e.g. Vector Labs SA-S100)
·  BCIP/NBT alkaline phosphatase kit (e.g. Vector Labs SK-5400)
·  100mM Tris -Hcl, pH9.5 for color development
·  35% ethanol (v/v in distilled water)
·  Sterile phosphate buffered saline (PBS)
·  Sterile distilled water
·  Complete R10 medium (RPMI-1640 supplemented with 10mM HEPES, 50mM 2-mercapto-ethanol, 100U/ml Penicillin,0.1mg/ml streptomycin, 2mM L-Glutamine and 10% fetal calf serum)
·  96 well PVDF membrane ELISPOT plates (Millipore #MSIPS4510)

Recommended Experimental Controls
·  Negative Control – no antigen stimulation / stimulation with known negative peptide
·  Positive Control – Stimulation with PHA

Protocol applicable for IL-2 ELISPOT assay
Aseptic Procedures (Use sterile buffers and aseptic conditions; use laminar flow hood for procedures)
1. Pre-wet plate wells with 15ml 35% ethanol for 1 minute. Aspirate, then wash twice with 200ml dH2O before the ethanol evaporates.
Once the membrane is pre-wet with alcohol, do not allow membrane to dry for the duration of the assay.
2. Dilute anti-IL-2 capture antibody to 15mg/ml in sterile PBS. Coat plate with 50ml/well. Incubate at 4°C overnight.
3. Decant or aspirate coating antibody from plate.
4. Wash plates 5 times with 300 ml/well sterile PBS. Decant.
5. Block plate with 200 ml/well of complete R10 medium at room temperature for at least 1 hour. Decant or aspirate plate.
6. Aliquot up to 300,000 cells/well in 100 ml complete R10. Aliquot peptide ([5mM] final) or controls diluted in complete R10 medium to appropriate wells.
The desired cell concentration depends on the intensity of the immune response. If the expected response is unknown, then we recommend a serial dilution of cell concentrations.
7. Incubate for 23-36 hours at 37°C, 5% CO2 and 95% humidity.

Incubation times should be developed and evaluated by the user. However, antigen-specific stimulation of cells results in detectable spots within 23 hours.

Non-Aseptic Procedures
8. Decant cells and medium from plates. Wash plate 5 times with 300ml PBS.
9. Dilute biotin-conjugated anti-Il-2 detection antibody in PBS and filter. Add 50ml/well to plate and incubate at room temperature for 3 hours.
Failure to filter the detection antibody may result in non-specific spot formation due to protein aggregates.
10. Decant antibody solution. Wash 5 times with 300ml PBS. Allow wells to soak for 1 minute for each wash.
11. Dilute alkaline-phosphatase reagent in PBS (final concentration 1mg/ml). Add 50 ml to each well and incubate at room temperature for 1 hour.
Exceeding 1 hour incubation with enzyme conjugate will result in increased background.
12. Discard solution. Wash plate 5 times with 300ml PBS.
13. Add NBT/BCIP reagent as per the manufacturers instructions and leave the substrate to develop until spots are clearly visible (approximately 30 minutes)
Optimization of substrate development time is critical, since over-development will result in increased background.
14. Stop the substrate development by washing wells 3 times with dH20.
15. Remove the under-drain from the plate and allow to air-dry.
16. Count spots by eye, or using an automated ELISPOT plate reader.

anti-HA antibody Western Blotting Protocol

1) Run gel (in 1:10 running/transfer buffer(10x) and H₂O for a total of 1litre) at 150 volts until leading bromophenol blue band is nearing the bottom edge of the glass plates or according to the expected migration of the protein of interest.

2) Transfer proteins in acrylamide gel to nylon membrane.

3) Block overnight @4℃ in PBS (or TBS, it doesn't matter) with 5% non-fat dry milk. I dilute 2.5 mg in 50 ml.

4) Wash off excess milk sol'n with PBS x3 .(important if you are re-using your antibody)

5) Incubate in primary antibody solution: I use PBS with 0.1% TWEEN-20 plus 5% BSA as the basic solution, but you can use TBS too. I dilute the HA antibody 1:5000, just to be sure you will see a signal. I haven't EVER seen appreciable background. I've also used it at 1:10K without any appreciable loss of signal. I usually go 30-90 minutes at RT (you can save the 1ab solution and use for a month or so).

6) Wash with 12-15 quick washes with PBS/Tween ( I literally pour it in, shake it around for 5-10 seconds, and pour it off: this method was worked out in the Yamamoto lab eons ago).

7) Incubate with secondary antibody (anti-mouse) in 5% BSA/PBS/Tween as you would ordinarily. I usually go for 2 hours @ room temp.

8) 12-15 quick washes again(in 1XPBS).

9) Assay for chemiluminescence or whatever you want. The normal procedure is to submerge blot in 20ml of ECL reagent for 2 minutes. Then wrap blot in saran wrap and wipe away excess ECLreagent. Place blot in film cartridge and bring to dark room. Expose film for ~2 mins.

Notes on the primary antibody:

Source : Covance/Babco

Description: Crude ascites fluid monoclonal anti-HA antibody (CATLN#135032002, 11mms-101R, 0.5 ml)

Handling: Aliquoted into 10ul portions in -20℃

Internal notes: labelled anti- Ha (Marc’s 1:5000)

Far Western Protocol

1. Run samples out on a gel. For bacterially expressed proteins, generally 5 μl is plenty (1ml cell cμlture; cells resuspeded in 50 μl loading buffer). Run the gels (BioRad mini gels) at 195 V for approximately 40 min. (until samples run close to the bottom of the gel).

2. Transfer the proteins from the gel onto nitrocellμlose. For the BioRad setup, the case shoμld be set up as follows: black side down, then 3M Scotch Brite Pad, then blotting paper, gel, nitrocellμlose, 2^nd blotting paper, 2^nd Scotch Brite Pad, and the clear side of the case. Put the case in the holder, black side of the case facing black side of the holder. Run the transfer at 100V for 1 hour.

3. Meanwhile, prepare 500 ml of AC Buffer (+ Tween):

50 ml glycerol (= 10% glycerol)

10 ml 5M NaCl (= 100mM NaCl)

10 ml1M Tris, pH 7.6 (= 20 mM Tris)

1 ml 0.5M EDTA (= 0.5mM EDTA)

5 ml 10% Tween-20 (= 0.1% Tween-20)

put on ice

b) Make 50 ml (or more) of 2% milk powder solution:

50 ml AC Buffer

1 g milk powder

-put on a rocker to dissolve the milk powder (may take 20-30 minutes). Then put on ice

4. Make the probe, using the TnT (Promega) Reticμlocyte Lysate kit.

Set up either a 25 μl reaction or a 50 μl reaction, depending on the size of tray you'll be using for washes and probing. Below is the recipe for a 50 μl reaction mix:

25 μl Reticμlocyte lysate (I use a little more, ~27 μl)

2 μl Reaction Buffer

1 μl T7 (or T3) polymerase (or other polymerase)

1 μl amino acids minus methionine (or missing other amino acid)

1 μl RNA Guard

4 μl 35S-met (or other labelled amino acid)

16 μl DNA + ddH₂O (1-2 μg DNA)

Spin down the sample (pμlse spin) to remove air bubbles. Let the reaction proceed at room temperature for 1 hour, 10 minutes (can be longer or shorter, depending on the protein).

Block, Probe, and Wash

5. After the transfer is complete, put the blot into a tray. Keep the side that was touching the gel up). Do 1-2 quick washes with 1X PBS to remove the SDS. Then, do a quick rinse in AC Buffer, to remove the PBS. Pour on the 2% milk powder (just enough to cover the blot), put a lid on the dish, and rock the blot at 4℃ for 1 hour. This is the blocking step.

6. Meanwhile, set up 2 spin columns:

Use 1cc syringes, remove the cap and the plunger. Put in glass wool to fill the opening at the bottom, push down with the plunger. Put the syringe in a 15 mL falcon tube. Add BioRad G 25 resin, stored at 4℃ in TE buffer, to the syringe , filling to the top (avoid air bubbles!). Spin down at 2000 rpm for 3 minutes. Then refill with G-25, and repeat the spin cycle. (At this point, the G-25 shoμld be packed down such that it occupies about 0.8ml.

Add to the columns 100 μl AC Buffer, spin at 2000 rpm for 3 min. Repeat two more times.

7. When the probe labeling reaction is finished, dilute the sample using AC Buffer to make a final volume of 100 μl. Load this on the spin column and spin at 2000 rpm for 3 minutes in a fresh falcon tube. Collect the flow-through. This process removes unincorporated nucleotides.

8. When there are approximately 20 minutes left in the blocking step, it is time to prepare the probe mix. At this point, you may want to collect a 2μl sample of your probe (from the 100 μl), and combine it with 10 μl 1X SDS gel loading buffer. You can then run this sample on a gel, and put film on the dried gel to see if the probe labelling step actually worked.

The volume of the probe mix depends on your initial probe reaction mix volume, and the size of tray that you are using. For example, if you set up a 50 μl initial reaction, you can use up to 20 ml of probe mix. For a 25 μl reaction, use no more than 10 ml of probe mix.

RNA isolation and quantification Protocol

The acid-phenol extraction is a simple procedure to extract RNA from cells, and to separate RNA from DNA. When cells are lysed and extracted with phenol, protein and lipids always partition into the phenol layer whereas RNA partitions to the aqueous layer (regardless of the pH of the phenol). In contrast, DNA shows a pH-dependent partitioning. When the aqueous phase of the phenol is buffered above pH 7.5, DNA partitions with RNA into the aqueous layer. However, when the aqueous phase of the phenol is buffered at pH 4.0, DNA will partition into the phenol layer. The concentration of RNA can be conveniently measured due to its ability to absorb light at 260 nm. The ratio A₂₆₀:A₂₈₀ is an indication of the purity of the RNA. The ratio (for pure RNA) should be 1.8 -2.1. However, even if the ratio is less than 1.8, the RNA quality may be acceptable and can be confirmed by the subsequent gel electrophoresis.

Materials required:
TRIzol Reagent (Gibco BRL; DNA and protein can also be extracted from the samples using this reagent)
Chloroform       Isopropanol        75% ethanol         RNase-free water (i.e. DEPC treated)
 

Phenol - Caution Phenol is highly toxic, even in small amounts. If spilt on skin - wash immediately under cold water If spilt on clothing - remove immediately If spilt on bench, mop up immediately and consult a demonstrator. UV light -Caution Spectrophotometers are a source of UV radiation

RNA isolation
1. Discard the cell growth medium safely.
2. Wash the cell monolayer by carefully by adding 1 ml PBS to the side of the dish making sure you do not disrupt the cells. Pour off the PBS wash.
3. Add 1 ml TRIzol to the plate to lyse the cells. Pass the cell lysate several times through a pipette (insufficient TRIzol may result in contamination of the isolated RNA with DNA).
4. Transfer the lysate to a 1.5 or 2.0ml Eppendorf tube.
5. Incubate the homogenized samples for 5 min at room temperature (this allows complete dissociation of nucleoprotein complexes).
6. Add 0.2 ml of chloroform per 1 ml of TRIzol (Make sure the tube is tightly capped).
7. Shake tubes vigorously by hand for 15 sec and incubate at room temp for 2.5 min.

RNA/DNA separation
1. Centrifuge samples at no more than 12,000 rpm for 15 min at 4℃. Following centrifugation the mixture separates into a lower, phenol-chloroform phase (red), an interface, and an aqueous upper phase (colourless) Ð RNA remains exclusively in the aqueous phase.
2. Transfer the aqueous phase (i.e. top layer; »600 µl) to a fresh tube.
3. To precipitate RNA, add 1 ml isopropanol per 1 ml of TRIzol used for the initial lysis and mix by inversion, then incubate at room temp for 10 min.
4. Centrifuge at 12,000 rpm for 10 min at 4ûC (cold-room)
5. Discard the supernatant and wash the RNA pellet with 75% ethanol (use at least 1 ml of 75% ethanol per 1 ml of TRIzol used for the initial lysis) - mix by vortexing.
6. Centrifuge at 7,500 rpm for 5 min at 4℃ (cold-room).
7. Discard the supernatant and (briefly) air-dry the RNA pellet by inverting the Eppendorf tube for 5-10 min. It is important NOT to dry the RNA completely.
8. Dissolve RNA in about 20ml RNase-free water
9. Heat RNA at 55 ûC for 5-10 min (increases solubility of RNA). Store at -20℃ o/n.

RNA concentration measurement
1. Dilute 2 - 3 µl of RNA (e.g. approx. 1/10 of RNA from above) into 500µl H₂O.
2. Place in a quartz cuvette and measure the OD₂₆₀ and OD₂₈₀.
3. Calculate the RNA concentration using the following equation:

Appendix

Concentration measurement
RNA (and DNA) has an absorbance peak at 260nm. The ratio A₂₆₀:A₂₈₀ is an indication of the purity of the RNA. The ratio (for pure RNA) should be 1.8 - 2.1. However, even if the ratio is less than 1.8, the RNA quality may be acceptable and can be confirmed by the subsequent gel electrophoresis.
Alternatives
Trizol can be purchased from GibcoBRL. If you wish to avoid phenol, many companies sell RNA extraction kits (e.g. Qiagen, Hybaid, etc).

Handling RNA -the hazards of RNase
One of the major difficulties in working with RNA is its susceptibility to hydrolytic cleavage by the enzyme ribonuclease (RNase). RNase is literally everywhere - it is secreted from the skin and tends to coat tubes, benches, pipettes an so on. It is also very stable Ð it retains its activity even after autoclave procedures and cannot be removed by cleaning surfaces with ethanol or isopropanol. It even retains activity after exposure to chaotropic agents such as guanidium thiocyanate. The only way to eradicate the effect of RNase is to chemically inactivate it. This requires the use of the methylating agent, diethylpyrocarbonate (DEPC), which irreversibly modifies the active site histidine residue of the enzyme, thereby inactivating it. All water used for RNA work must be treated with DEPC before use. DEPC is dispensed in a fume hood and added to water in an autoclavable (Schott type) bottle containing a stir bar. The water is stirred for one hour in the fume cupboard and after treatment the solution is autoclaved to denature the DEPC. To avoid contamination by RNase, it is important to handle solutions and material for RNA use with extra care:
-Wear gloves at all times
-Maintain separate stocks of chemicals reagents for RNA work - do not use for other laboratory stocks. Many suppliers can provide RNase-free chemicals and reagents.
-Solutions that have been prepared for RNA work should be kept separate from the rest of the laboratory solutions- label these reagents appropriately.
-All pipette tips and Eppendorf tubes should not be handled by hand prior to autoclaving. If possible, purchase pre-packed RNase free materials.

First-strand cDNA Synthesis

Prepare in a sterile tube:

template RNA:

total RNA  0.1-5µg

or poly(A)+ mRNA  10ng-0.5µg,

or specific RNA  0.01pg-0.5µg

primer:

oligo(dT)18   0.5µg

or random hexamer 0.2µg,

or sequence-specific 15-20pmol,

deionized water (nuclease free) up to 11µl.

Incubate the mix at 70℃for 5 minutes and chill on ice.

Add the following in the order indicated:

5X reaction buffer 4µl,

10mM 4 dNTP mix 2µl (1.0mM - final concentration),

ribonuclease inhibitor 20u,

deionized water (nuclease free) to 19µl.

Incubate at 37℃for 5 minutes. If random primer is used, incubate at 25℃for 5 minutes.

Add 40 units of M-MuLV Reverse Transcriptase . Incubate the reaction mixture, containing oligo(dT)18 or sequence-specific primer at 37℃for 60 minutes. If using random hexamer primer, incubate at 25℃for 10 minutes and then at 37℃for 60 minutes.

Stop the reaction by heating at 70℃for 10 minutes. Chill on ice.

Note

The synthesized cDNA can be amplified by the PCR (see Protocols for PCR using Taq and Pfu DNA Polymerases) without intermediate phenol/chloroform extraction or ethanol precipitation.

Reference

Gerard, G.F. and D'Alessio, I.M., Methods in Molecular Biology, 16, Humana Press, Totowa, N.J., 73-93, 1993.

Intracellular Cytokine Staining Protocol

Intracellular Staining:
7. Resuspend fixed/permeabilized cells in residual Permeabilization Wash
Buffer and add a predetermined optimum concentration of fluorochrome
conjugated antibody of interest (e.g. anti-IFN-γ-PE) or an appropriate negative
control for 20 minutes in the dark at room temperature.
8. Wash 2x with 2 ml of Permeabilization Wash Buffer and centrifuge at 350 x g
for 5 minutes.
9. If primary intracellular antibody is biotinylated, it will be necessary to perform
fluorochrome conjugated Streptavidin incubations and subsequent
washes in Permeabilization Wash Buffer.
10. Resuspend fixed and intracellularly labeled cells in 0.5 ml Cell Staining Buffer
and analyze with appropriate controls.
Note: To confirm specific anti-cytokine staining, a blocking experiment is recommended
in which cells are fixed/permeabilized then preincubated with an
excess amount of unlabeled anti-cytokine antibody and/or the recombinant
cytokine of interest is preincubated with fluorochrome-conjugated anticytokine
antibody before its addition to the cells.

Cellular ELISA Protocol

A. Formalin Fixed Cell Plates

1. Trypsinize confluent flasks

2. Pool and count cells

3. Centrifuge at 1500 rpm for 10 minutes

4. Resuspend to the appropriate concentration in complete medium
4 x 10⁵ cells/ml for epithelial cells
2 x 10⁵ cells/ml for fibroblast cells

5. Add 100 ml/cell to 96 well culture plates.

6. Incubate overnight at 37^oC.

7. Wash plates twice with PBS

8. Add 125 ml/well 10% Buffered Formalin

9. Fix for 15 minutes at room temperature

10. Wash three times with di-H₂O.

11. Blot dry.

12. Store at 2-8^oC.

B. Reagents

1. PBS:1% BSA

2. PBS:2% BSA

3. Carbonate Buffer
1.59 g  Na₂CO₃
2.93 g  NaHCO₃
Dissolve in 900 ml di-H₂O. Check pH and adjust to 9.6 necessary. Qs. to 1 liter.

4. 10X Substrate Buffer, pH 6.0
36.6 g  Citric Acid, monohydrate
113.5 g  Potassium dibasic phosphate
Dissolve in 900 ml di-H₂O. Check pH and adjust to 6.0 if necessary. Qs. to 1 liter.

5. 0.3% H₂O₂
Dilute 30% stock Peroxide 1:100 in di-H₂O.

6. OPD Stock, 4.0%
4 g OPD in 100 ml di-H₂O.  Aliquot and store at -20^oC. Protect from light.

7. 4.5N H₂SO₄
12.0 ml  Concentrated Sulfuric Acid
88.0 ml  di-H₂O

C. Procedure

1. Wash ELISA plates once with di-H₂O.

2. Add 250 ml/well PBS:2% BSA.

3. Incubate 1 hour at 37^oC.

4. Wash 3 times with di-H₂O.

5. Add 50 ml/well supe, ascites, or controls diluted in PBS:1%BSA.

6. Incubate for 2 hr at 37^oC.

7. Wash 5 times with di-H₂O.

8. Add 50 ml/well anti-mouse IgG:HRP diluted in PBS:1% BSA.

9. Incubate for 1 hr at 37^oC.

10. Wash 5 times with di-H₂O. Wash once with carbonate buffer.

11. Add 50 ml/well working substrate solution
0.5 ml  4.0% OPD
5 ml  30% H₂O₂
1.0 ml  10X Substrate buffer
8.5 ml  di-H₂O.

12. Incubate for 20 minutes at room temperature.

13. Add 25 ml/well 4.5N Sulfuric Acid

14. Read A₄₉₀

D. Notes

1. Test all supernatants at 1:5 dilution.

2. Test ascites at 1:100

Gangliosides ELISA protocol

A. Abstract

This protocol can be used for detection of gangliosides.Spicific antibodies to gangliosides are added to cells and allowed to bind to the cell surface. The nonbound antibody is washed away and an enzyme conjugated second antibody is then detected by the addition of an enzyme-spicific substrate.

B. Overview

This protocol can be used for detection of gangliosides.Spicific antibodies to gangliosides are added to cells and allowed to bind to the cell surface. The nonbound antibody is washed away and an enzyme conjugated second antibody is then detected by the addition of an enzyme-spicific substrate.

C. Procedure

1. Trypsinize confluent flasks

2. pool and count cells

3. Resuspend to 4x100,000 cells/ml medium.

4. Add100ul/cell to 96 well culture plates

5. Incubate 24Hrs at 37c

6. Wash plates twice with PBS

7. add 150ul/well 10% Buffered Formalin

8. Fix for 20 minutes at room temperature

9. Wash three times with PBS

10. Add 250ul/well PBS:2%BSA

11. Incubate 30minutes at room temperature.

12. Wash plates twice with PBS.

13. Add 100ul/well primary antibody diluted in PBS

14. incubate for 2hr at 37c

15. Wash 5 times with PBS.

16. Add 50ul/well secondary antibody:HRP diluted in PBS.

17. Incubate for 1hr at 37c.

18. Wash 5 times with PBS.

19. TMB stoped 450nm.

Peptide Inhibition ELISA Protocol

A. Reagents

PBS

 PBS:0.1% BSA:0.05% Tween 20 (PBT)

PBS:2% BSA

10X Substrate Buffer, pH 6.0
36.6 g      Citric Acid, monohydrate
113.5 g      Potassium dibasic phosphate
Dissolve in 900 ml di-H₂O. Check pH and adjust to 6.0 if necessary. Qs. to 1 liter.

30% H₂O₂

OPD Stock, 4.0%
4 g OPD in 100 ml di-H₂O. Aliquot and store at -20^oC. Protect from light.

4.5N H₂SO₄
12.0 ml      Concentrated Sulfuric Acid
88.0 ml      di-H₂O

B. Procedure

Titer MAb supes at serial 5-fold dilutions to determine the concentration at which the titer begins to drop.

 Dilute the MAb to one-half the above dilution in PBT

Dilute specific and non-specific peptides to 200 mg/ml in PBT.

In a microtiter plate, mix equal volumes of the peptide dilutions and the MAb supe. Add an equal volume of PBT and MAb as the control.

 Incubate 1 hour at 37^oC.

Transfer 50 ml to an antigen coated plates, which has been blocked with PBS:2% BSA for 1 hour at 37^oC.

 Incubate for 1 hour at 37^oC.

Wash 3 times with di-H₂O.

Add 50 ml/well anti-mouse IgG:HRP diluted in PBS:0.1% BSA:0.05% Tween 20.

Incubate for 1 hr at 37^oC.

Wash 5 times with di-H₂O.

Add 50 ml/well working substrate solution
0.5 ml      4.0% OPD
50 ml      30% H₂O₂
1.0 ml      10X Substrate buffer
8.5 ml      di-H₂O.

Incubate for 20 minutes at room temperature.

Add 25 ml/well 4.5N Sulfuric Acid

Read A₄₉₀

T7 siRNA protocol

Here is our T7 siRNA protocol. The main idea is to design primers that begin with GG so that

transcription by T7 polymerase is efficient.

I) FIRST design oligos:

Using sense coding sequence of any gene....

(N1, N2, N22, N23, are numbered positions)

1) Find 5'-N1 N2 G/A G/A NNNNNNNNNNNNNNN C C N22 N23-3'

2) Drop N22 and N23

add 5'-TATAGTGAGTCGTATTA-3' to 3' END to get 5'-N1 N2 G/A G/A NNNNNNNNNNNNNNN C C TATAGTGAGTCGTATTA-3' = oligo A

3) From (1) 5'-N1 N2 G/A G/A NNNNNNNNNNNNNNN C C N22 N23-3' drop N1 N2 convert G/A G/A to G G to get 5'-G G NNNNNNNNNNNNNNN C C N22 N23-3'

4) Add 5'-TAATACGACTCACTATA-3' to 5'END to get 5'-TAATACGACTCACTATA G G NNNNNNNNNNNNNNN C C N22 N23-3'

5) Get reverse complement of (4) 5'-N23' N22' G G NNNNNNNNNNNNNNN' C C TATAGTGAGTCGTATTA-3' = oligo B

6) Rank the initial target sequences with GG preferable to G G/A preferable to G/A G with A A not even considered unless absolutely necessary.

GG N15 CC > G G/A N15 CC > G/A G N15 CC >>>>>>>>> A A N15 CC

7) Order oligos

II) THEN make RNA in vitro

8) Anneal oligo A and oligo B separately to T7 primer (TAATACGACTCACTATAGG) or toprimers that are complementary to oligoA and B.

9) Use 2-3 ug annealed primer in a 20ul Ambion T7 Megashort script reaction Ambion Cat# 1354 (Follow Ambion’s protocol Incubate 2-4 hrs)

10) Combine oligo A and B reactions

11) Anneal T7 transcribed RNAs using your favorite slow annealing protocol

e.g.

95℃ 5 min

70℃ 5min

50℃ 5min

37℃ 5min

12 A) Phenol-chloroform extract annealed, transcribed RNAs / Ethanol precipitate /

12 B) Or Purify on Ambion MegaClear columns

13) Resuspend in 50-250ul H2O

14) Quantify yield

15) Transfect

use at least 0.5ug per well of a 6 well plate (3.0ug per 10 cm dish)

Western Blotting Protocol

Buffers:

TBS:  25 ml of 1 M Tris-7.5, 30 ml of 5 M NaCl, bring volume up to 1000 ml with ddwater

TBS-T:  TBS + 0.5 ml of Tween 20

5X SDS-PAGE running buffer:  15.1 g Tris base, 94 g glycine in 900 ml water, then add 50 ml of 10% solution of SDS, and adjust volume to 1000 ml with ddwater

Blocking solution:  5 g nonfat dry milk in 100 ml of PBS

Transfer Buffer:  5.82 g Tris base + 2.93 g glycine + 0.375 g SDS (or 3.75 ml of a 10% solution of SDS) + 200 ml methanol, then bring volume up to 1 liter with ddwater

Prepare cell extracts using an appropriate protocol.

1. Measure total protein in extract by the Bradford method.  Plan on loading approximately 5 μg of protein per lane on the polyacrylamide gel.

2. Add an equal volume of SDS sample buffer and boil the sample on a 100℃ block for 5 min.

3. Apply the sample in about 10 μl volume to an appropriate percentage (typically 4-20% gradient) SDS-PAGE mini gel, along with visible protein marker (5 ul) and appropriate positive and negative controls.  Run gel using 1X SDS running buffer.

4. Run gel at 25 mA (constant) for 30 min to 1 hr. (or until blue dye just runs off of the gel)

5. Transfer onto immobilon P membrane using the SD semi-dry transfer apparatus, per apparatus instructions.

6. After transfer, ensure that all the protein has transferred, as assessed by transfer of the visible protein marker onto the immobilon P membrane.

7. Block in 75 ml of blocking solution overnight at 4 ℃(alternatively, block at room temp for 1 h on a shaking platform).

8. Wash 3 times with TBS-T, each for 10 min.

9.Make dilution of primary antibody (usually 2 μl in 10 ml of TBS), and add to membrane, shake on a platform for 1 h at room temp. 

Make sure that membrane is entirely covered with antibody solution.

10.  Discard antibody solution and wash with TBS-T 3 times for 10 min each.

11.  Make dilution of secondary antibody (usually 1 μl in 10 ml), and cover membrane with antibody solution and shake on a platform for 1 h at room temp.

12.  Discard antibody solution, and wash membrane with TBS-T 4 times for 10 min. each.

13.  Detection using the ECL/ECLplus kit.