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.

DUAL ELISPOT PROTOCOL

staining provides the opportunity to detect two target proteins at the same time in the ELISPOT cell assay. An advantage of this is that it enables the researcher to detect a marker protein to determine which cell type is secreting the protein, as well as the secreted protein of interest.
The following dual ELISPOT procedure describes a protocol using an FITC-conjugated primary antibody and a biotinylated primary antibody. Those are in turn recognized by anti-FITC HRP and streptavidin-AP conjugates. PVDF-bottomed-well plates are then incubated first with AEC substrate buffer, washed and subsequently incubated with BCIP/NBT. The colored spots will either be red/brownish blue/purple so the two target proteins can be distinguished.

Materials and Reagents:
1.Detection antibody
2.Streptavidin alkaline phosphatase conjugate
3. Phosphate buffered saline (10 x Concentrate solution).
For 1 liter weight : 80g NaCl ; 2g KH2PO4; 14.4g Na2HPO42H2O. Add distilled water to 1 liter. Check that pH is 7.4+/- 0.1. Dilute the solution to 1X before use.
4. 2% dry skimmed milk in PBS
For one plate dissolve 0.2 g of powder in 10 mL of 1X diluted PBS.
5. 1% BSA in PBS
For one plate dissolve 0.2 g of BSA in 20 mL of 1X diluted PBS.
6. 0.1% Tween in PBS
For one plate dissolve 100􀀁l of Tween 20 in 100 ml of 1X diluted PBS.
7. 70% ethanol in water
For one plate mix 7 ml of ethanol with 3 ml of distilled water.
8. AEC Buffer
For one plate mix 1 ml of ACE Buffer A with 9ml of distilled water. Then add 200􀀁l of ACE Buffer B.
4. ELISPOT 96 well plates
(provided with Abcam ELISPOT kits)

AFLP Protocol

Restriction digestion
Master mix preparation:
Prepare a master mix of the following per sample, plus 5 to 10% extra to allow for pipetting loss.
5X R/L buffer (see page 4 for recipe) 6.0 μl
EcoR I (12 U @20 U/μl) 0.6 μl
Mse I (8 U @ 4 U/μl) 2.0 μl
Water to 10 μl
Reaction time:
Incubate for at least 1 hr. at 37°C, but not longer than 3 hrs, before proceeding with the ligation.

Adapter ligation
Master mix preparation
If 10 μl of the restriction reaction was removed for gel analysis, prepare a ligation master mix of the following, per sample, plus enough for 5-10 extra samples:
EcoRI adapter (@ 5 pMol/μl) 0.5 μl
MseI adapter (@ 50 pMol/μl) 0.5 μl
ATP (10 mM, pH 8.0) 0.5 μl
5X R/L buffer 1.0 μl
sdH2O 2.0 μl
T4 DNA ligase (0.5 Weiss U @ 1 U/μl) 0.5 μl
TOTAL 5.0 μl
Reaction time:
Incubate for at least 3 hrs. (preferably overnight) @ 37° C. After incubation, dilute each R/L mix 1:10 with sdH₂O.

Preamplification
Master mix preparation
Prepare a master mix with the following amounts per sample, plus 5-10 samples extra:
10X PCR buffer 3.0 μl
dNTP mixture (2.5 mM ea.) 2.4 μl
E primer (@ 50 ng/μl ≅ 8.3μM) 1.0 μl
M primer (@ 50 ng/μl ≅ 8.3μM) 1.0 μl
Taq polymerase (@ 5 U/μl) 0.4 μl
sdH₂O 19.2 μl
μl per sample 27.0 μl

Important: If pre-amplifications are to be done in 96-well PVC plates instead of polycarbonate or polypropylene plates or tubes, it may be necessary to add 2.0 μl of 10 μg/μl BSA per sample to the Taq-buffer mix, and decrease the amount of H2O to 5.76 μl per sample. DO NOT use a hot start PCR protocol, or a hot start polymerase (e.g. AmpliTaq Gold), for the pre-amplification! The adapters are non-phosphorylated, so only the top strand is ligated. The bottom strand of the adapter will separate from the rest of the template first and must be re-synthesized during the initial heating stage. This requires polymerase activity during the initial heating.
PCR program
Place reactions in the thermocycler, and run the following PCR amplification profile:
28 cycles: 15 sec @ 94°C denaturation
30 sec @ 60°C annealing
60 sec + 1sec/cycle @ 72°C extension
1 cycle: 2 min @ 72°C final extension
hold: 4°C

Final AmplificationMaster mix preparation (single dye reactions)
For each E/M primer combination to be used, prepare the following master mix, per sample, in an Eppendorf tube, plus enough for 5-10 samples extra:
10X PCR buffer 2.0 μl
dNTP mixture (2.5 mM ea) 1.6 μl
IRD-labeled E-primer (@ 6 ng/μl ≅ 1μM) 0.83 μl
M-primer (@ 50 ng/μl ≅ 8.3μM) 0.6 μl
Taq polymerase (@ 5U/μl) 0.24 μl
dH₂O 9.73 μl
Final volume 15.0 μl

Cleaved Amplified Polymorphic Sequences (CAPS)

Cleaved Amplified Polymorphic Sequences (CAPS) polymorphisms are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonucleaserecognition sites in PCR amplicons produced by locus-specific oligonucleotide primers.

How It Works

The CAPS assay uses amplified DNA fragments that are digested with a restriction endonuclease to display RFLP.

Unique sequence primers are used to amplify a mapped DNA sequence from two related individuals (for example, from two different inbred ecotypes), A/A and B/B, and from the heterozygote A/B. The amplified fragments from A/A and B/B contain two and three RE recognition sites, respectively. In the case of the heterozygote A/B, two different PCR products will be obtained, one which is cleaved three times and one which is cleaved twice. When fractionated by agarose or acrylamide gel electrophoresis, the PCR products digested by the RE will give readily distinguishable patterns. Some bands will appear as doublets.

Advantages

• Most CAPS markers are co-dominant and locus-specific.
• Most CAPS genotypes are easily scored and interpreted.
• CAPS markers are easily shared between laboratories.
• CAPS assay does not require the use of radioactive isotopes, and it is more amenable, therefore, to analyses in clinical settings.

Developing CAPS markers

• Sequence the RFLP probe.
• Design primers to amplify 800–2,000-bp DNA fragments. Targeting introns or 3' untranslated regions should increase the chance of finding polymorphisms
• The PCR product is cloned and sequenced.
• PCR amplify DNA fragments from target genotypes, separately digest the amplicons with one or more restriction emzymes.
• Screen the digested amplicons for polymorphism on gels stained with ethidium bromide.

QRT-PCR protocol

General notes:

       Consistency is crucial to the accuracy of QRT-PCR, so the extra steps in this protocol are necessary. In general, be extra careful to treat every sample the same. Always use filter tips, and do no aspirate when pipetting (only go down to the first stop). Avoid bubble formation, which can accelerate the degradation of the cDNA. The SYBR Green is photosensitive, so the mixing is done in the “dark” room in Judy Manning’s lab, which is also the location of the iCycler machine. For the final spin of the 96-well plate, wrap the plate in foil before bringing it into the lab, and minimize exposure to light. Samples are done in triplicate to ensure that the technique is good.

Before beginning protocol

1. Determine what templates will be used, and which will be used in the dilution series. At least one template should have a dilution series of five 10-fold dilutions.
2. For the primer set being used, determine the best annealing temperature by doing end-point PCR using a gradient for the annealing temperature. Pick the highest temperature with the best band. Use this temperature for the QRT-PCR reaction. Because of this, different primer sets must be done in separate iCycler runs, unless the best annealing temperature is the same for two sets.
3. Sign up for the iCycler ahead of time. The sign-up sheet is currently at the end of the McFall-Ngai lab bench on the left side of the aisle, as you face the windows.
4. It is a good idea to get the iCycler program and well designations done before starting work with the samples.

Protocol:

1. Make master mix: Multiply the volumes per reaction by the number of reactions, and add 10%.

       Component                                Volume added per reaction

       2x iQ SYBR green                        12.5 ul

       Forward Primer (10 uM)               0.5 ul   

       Reverse Primer (10 uM)                   0.5 ul

       Sterile Distilled H₂O                            10.5 ul

      

       Total volume                               24.0 ul

Note: the amount of primers used will vary with the stock concentration. This assumes that 1 ul template is added per reaction. If this is not the case, adjust the volumes accordingly.

2. Vortex mix briefly, then do a short spin to remove any bubbles.

3. Prepare PCR tubes with 3 ul of each template.

5. Add 72 ul of master mix to each tube.

6. Vortex and spin tubes briefly.

7. Place 23 ul of each sample in the appropriate wells in a 96-well plate (less than the full amount is used to make sure that there is enough for all three wells, and to avoid bubbles). Make sure to put the samples in the correct well, as only those wells designated by the program will be read and recorded.

8. Carefully put clear cover on the plate. Use paddle to smooth down – do not touch cover.

9. Wrap plate in foil. Find similarly filled plate (there’s probably a pile) for a balance. Take into main lab, and spin down in Sorval Legend RT at *** for **** minutes.

10.  Return to iCycler room. Keep plate in cold block. Begin iCycler run, and when the block gets up to temperature, pause the program and place the plate in the block. Well A1 goes in the top left as you are facing the machine. Unpause the program, and allow to run. Do not turn on the lights in the room until the run is over.