Western Blotting Tips

1) Load 20 to 35 μl of your samples unless you know for a fact that this is too much.

2) In the first lane load 25 μl of positive bloting control. This is lysate stimulated with PMA or LPA. This is good for most CCL39 blots.

3) Molecular Wt Markers - There are now two kinds. One is the BioRad Prestained standards we've always used. The other is MagicMarker. This is a non-stained protein that has an IgG recognition site. That means each of the proteins will bind secondary antibody and will be visible on the film. To use this:

- In the second lane load 10-15 μl of the BioRad standard

- In the SAME well/lane add 3 μl of the MagicMarker standard (found in a box in the

freezer in the main room

4) Run the gel to the bottom but not off. Make certain the dye is just coming to the edge of the glass not the gasket. Remember, 200 v for 1 hour is an approximation - you may very likely have to turn it up a little more. If this is a key experiment, run the gel at 100 v for 10-15 min first then run for 200V. The proteins will be tighter. ALSO if the % cross-linking is low then the proteins will run faster than the higher % gels

5) Soak the gel in transfer buffer for a minute or two to reduce salt that can reduce transfer.

6) Cut the paper to fit the gel, unless you are using ERK or p-ERK. Remember unless we have done the blot many times you need to show the molecular weights to prove that the band you think is your protein is the right one. Clip the corner of the blot as directed in the protocol. Not all antibodies are as specific as some of the ones we use. Always mark the film with the markers!

7) The fit of the gel sandwich should be tight and free of air bubbles. If the pads are getting

thin add a third to make certain there is proper contact between paper and gel.

8) Transfer at 90 V for 1 hr unless using a 12-16% gel then add 15 min

9) Some antibodies need a different washing solution. The concentration or type of detergent (Tween-20) may not work for some. If using a new antibody, check!

10) Block in the BioRad dry milk, not the carnation or other generic instant milk. Use the same concentration as before. We get high backgrounds with some of these other milks. Some antibodies need BSA instead of milk to block with. Always look up new antibody requirements.

11) When starting use 1:500 dilutions for the primary and 1:1000 for the secondary. Use only the antibodies from the "Chinese take-out" cool-safe box

12) Don't let the blot get too dry - this may have been a problem

13) Do a 10 sec, 1 min and 10 min exposure.

Purifying DNA from TAE agarose gels

1. Excise band from agarose gel.

2. Add 3 volumes of NaI and incubate at 55°C to melt gel.

3. Add 5μl GLASSMILK suspension.

4. Pellet GLASSMILK/DNA complex (5 seconds).

5. Wash pellet with 1ml NEW Wash.

6. Pellet again and rewash with 0.5ml NEW Wash.

7. Elute DNA with 10-20μl water or 0.1x TE.

Proteolytic enzymes

Trypsin, collagenase, or pronase, usually in combination with EDTA, causes cells to detach from the growth surface. This method is fast and reliable but can damage the cell surface by digesting exposed cell surface proteins. The proteolysis reaction can be quickly terminated by the addition of complete medium containing serum.

Lipid siRNA transfection

For some cell lines, either electroporation or cationic lipids may be used. Basically any tfx protocol will require you to work out conditions for every cell line. Sometimes between 70-90% or more cells can be transfected with cationic lipids. With a bit of tweaking, and depending on the cell line, you can drive siRNAs into almost 100% of the cells with the following protocol:

Seed cells high, so that they are 85%+ on the day of transfection:

For one 10 cm dish:

40 ul Lipofectamine 2000

20 ug DNA

100-300 pmoles siRNA

tube 1

add the siRNA and/or DNA to 2 ml SF-DMEM (no drugs added)

tube 2

add the lipid to 2 ml SF-DMEM (no drugs added)

mix these two tubes immediately, for a volume of 4 ml, and then add to cells which have been washed 2X with SF-DMEM. Incubate 3-6 hours, then add normal media. Check for expression or knockdown 36+ hours post-transfection. Knockdown by siRNAs usually last 3-5 cell doublings.

When oligofectamine is used, the identical protocol is used, but:

Seed cells at 15% confluency, perform tfx with 30 ul oligofectamine and siRNAs, then repeat tfx 36 hours later. Analysis is performed when cells are 85-90% confluent. I have not calculated the transfection efficiency with oligofectamine.

ELISPOT Protocol

Coat the Plate:
1. Dilute Low-Endotoxin/Azide-Free sterile unlabeled capture antibody (BioLegend’s
LEAF™ format antibodies are specifically designed for this assay) to a
final concentration of 0.5–4 μg/ml in sterile Coating Buffer and transfer 100
μl/well to a high affinity binding PVDF membrane ELISPOT plate (e.g., Millipore;
Cat. No. MAIPS-4510).
2. Store plates overnight in humidified box at 4°C or at 37°C for ≥ 4 hours in
humidified atmosphere.

Block the Plate:
3. Wash plate 3 times with sterile PBS, 200 μl/well.
4. Add 200 μl/well of sterile Blocking Buffer.
5. Seal plate and incubate at room temperature for ≥ 1 hour.
6. Wash plate 3 times with sterile PBS, 200 μl/well.
Set-Up Tissue Culture and Add Antigen or Mitogen:
7. Add appropriate sterile antigen or mitogen solution diluted in appropriate
sterile tissue culture medium (TC) to ELISPOT plate, 100 μl/well.
8. Add cells diluted in sterile TC medium, 100 μl/well. Use 50,000-500,000 cells/
well (the minimum number of cells should be determined in preliminary
experiments).
9. Seal plate and incubate at 37°C 5% CO2 in humidified atmosphere for the
optimum stimulation period. BioLegend recommends a 24 hour incubation
for IFN-γ, IL-2, and TNF-α, and a 48 hour incubation period for IL-4, IL-5, and
IL-10 for most activation conditions.

Add Detection Antibody:
10. Wash plate 3 times with PBS, 200 μl/well.
11. Wash plate 3 times with PBS-Tween, 200 μl/well.
12. Add 100 μl/well of diluted biotinylated detection antibody at 0.25-2 μg/ml in
PBS-Tween-BSA.
13. Seal the plate and incubate at 4°C overnight, or 2 hr at room temperature.

Add Avidin-Horseradish Peroxidase (Av-HRP):
14. Wash plate 4 times with PBS-Tween, 200 μl/well.
15. Add 100 μl per well of the Av-HRP conjugate (Cat. No. 405103) or other
enzyme conjugate diluted to its pre-determined optimal concentration in
PBS-Tween-BSA (usually between 1/500 – 1/2000).

Source: 1. postech.ac.kr

2.http://www.biolegend.com/media_assets/support_protocol/BioLegend_ELISPOT_protocol.pdf

Competitive ELISA Protocols

AbVideo™ - Competitive ELISA

3 May 2010 ... Competitive ELISA is through competitive binding to measure the amount of analyte. First of all, unlabeled antibody is incubated in the ...
www.abnova.com › Support › AbVideo™

Millipore - Competitive ELISA assays, Competitive ELISA protocol.

Competitive ELISA assays, Competitive ELISA protocol and Competitive ELISA method - General Protocol for the Competitive ELISA Method.
www.millipore.com/cellbiology/.../competitiveelisamethod

Clinical utility of a competitive ELISA to detect antibodies ...
Clinical Utility of a Competitive ELISA. 85 lines for their use as a screening test. Following the comple- tion of the present evaluation, we proposed a ...
www.ugr.es/~cts521/documentos/Gutier-JClinLabAnal14.pdf

Competitive ELISA
In the competitive ELISA, two antigens are involved; one, the sample antigen to be assayed and the other, a constant level of "binding" antigen immobilized ...
www.springerlink.com/index/urh3412h3612g473.pdf

Competition Elisa Protocols | eHow.com

7 Dec 2010 ... Competition Elisa Protocols. The ELISA test is an enzyme-linked immunosorbent assay that uses antibodies or antigens coupled to a specific ...
www.ehow.com › ... › Science & Nature › Science

Springer Protocols: Competitive ELISA

2 Direct Competitive ELISA ... 2.1 Learning Principles; 2.2 Reaction Scheme; 2.3 Materials and Reagents; 2.4 Practical; 2.5 Competition Assay Prope. ...
www.springerprotocols.com/Abstract/doi/10.../0-89603-279-5:177

Competitive ELISA

Competitive ELISA is through competitive binding. The steps for this ELISA are somewhat different than the others:

1. Unlabeled antibody is incubated in the presence of its antigen (Sample)

2. These bound antibody/antigen complexes are then added to an antigen-coated well.

3. The plate is washed, so that unbound antibody is removed. (The more antigen in the sample, the less antibody will be able to bind to the antigen in the well, hence "competition.")

4. The secondary antibody, specific to the primary antibody is added. This second antibody is coupled to the enzyme.

5. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.

For competitive ELISA, the higher the sample antigen concentration, the weaker the eventual signal. The major advantage of a competitive ELISA is the ability to use crude or impure samples and still selectively bind any antigen that may be present.

(Note that some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with your sample antigen (unlabeled). The more antigen in the sample, the less labeled antigen is retained in the well and the weaker the signal).

Commonly the antigen is not first positioned in the well. (Wikipedia)

Competitive Elisa Protocol

1. For most applications, a polystyrene microtiter plate is best; however, consult manufacturer guidelines to determine the most appropriate type of plate for protein binding.

2. Add 50 µL of diluted primary antibody (capture) to each well. The appropriate dilution should be determined using a checkerboard titration prior to testing samples. PVC will bind approximately 100 ng/well (300 ng/cm2). The amount of antibody used will depend on the individual assay, but if maximal binding is required, use at least 1 µg/well. This is well above the capacity of the well, but the binding will occur more rapidly, and the binding solution can be saved and used again. Allow to incubate for 4 hours. at room temperature or 4°C overnight. Note: If a purified capture antibody is not available, the plate should first be coated with a purified secondary antibody directed against the host of the capture antibody according to the following procedure: (1) Bind the unlabeled secondary antibody to the bottom of each well by adding approximately 50 µL of antibody solution to each well (20 µg/mL in PBS). (2) Incubate the plate overnight at 4°C to allow complete binding. (3) Add primary capture antibody (as above).

3. Wash the wells twice with PBS. A 500 mL squirt bottle is convenient. The antibody solution washes can be removed by flicking the plate over a suitable container.

4. The remaining sites for protein binding on the microtiter plate must be saturated by incubating with blocking buffer. Fill the wells to the top with 3% BSA/PBS with 0.02% sodium azide. Incubate for 2 hrs to overnight in a humid atmosphere at room temperature.

5. Wash wells twice with PBS.

6. Add 50 µL of the standards or sample solution to the wells. All dilutions should be done in the blocking buffer (3% BSA/PBS with 0.05% Tween-20).

7. Note: Sodium azide is an inhibitor of horseradish peroxidase. Do not include sodium azide in buffers or wash solutions, if an HRP-labeled conjugate will be used for detection.

8. Add 50 µL of the antigen-conjugate solution to the wells (the antigen solution should be titrated). All dilutions should be done in the blocking buffer (3% BSA/PBS with 0.05% Tween-20). Incubate for at least 2 hours. at room temperature in a humid atmosphere.

9. Wash the plate four times with PBS

10. Add substrate as indicated by manufacturer. After suggested incubation time has elapsed, optical densities at target wavelengths can be measured on an ELISA reader.

11. Note: Competitive ELISAs yield an inverse curve, where higher values of antigen in the samples or standards yield a lower amount of color change.

(Source: Millipore)