LRRK2 Antibody - BSA Free

Protocol for Flow Cytometry Intracellular Staining
Sample Preparation.
1. Grow cells to 60-85% confluency. Flow cytometry requires between 2 x 105 and 1 x 106 cells for optimal performance.
2. If cells are adherent, harvest gently by washing once with staining buffer and then scraping. Avoid using trypsin as this can disrupt certain epitopes of interest. If enzymatic harvest is required, use Accutase, Collagenase, or TrypLE Express for a less damaging option.
3. Reserve 100 uL for counting, then transfer cell volume into a 50 mL conical tube and centrifuge for 8 minutes at 400 RCF.
a. Count cells using a hemocytometer and a 1:1 trypan blue exclusion stain to determine cell viability before starting the flow protocol. If cells appear blue, do not proceed.
4. Re-suspend cells to a concentration of 1 x 106 cells/mL in staining buffer (NBP2-26247).
5. Aliquot out 1 mL samples in accordance with your experimental samples.

Tip: When cell surface and intracellular staining are required in the same sample, it is advisable that the cell surface staining be performed first since the fixation and permeablization steps might reduce the availability of surface antigens.

Intracellular Staining.
Tip: When performing intracellular staining, it is important to use appropriate fixation and permeabilization reagents based upon the target and its subcellular location. Generally, our Intracellular Flow Assay Kit (NBP2-29450) is a good place to start as it contains an optimized combination of reagents for intracellular staining as well as an inhibitor of intracellular protein transport (necessary if staining secreted proteins). Certain targets may require more gentle or transient permeabilization protocols such as the commonly employed methanol or saponin-based methods.
Protocol for Cytoplasmic Targets:
Optional: Perform cell surface staining as described in the previous section.
1. Fix the cells by adding 100 uL fixation solution (such as 4% PFA) to each sample for 10-15 minutes.
2. Permeabilize cells by adding 100 uL of a permeabization buffer to every 1 x 106 cells present in the sample. Mix well and incubate at room temperature for 15 minutes.
a. For cytoplasmic targets, use a gentle permeabilization solution such as 1X PBS + 0.5% Saponin or 1X PBS + 0.5% Tween-20.
b. To maintain the permeabilized state throughout your experiment, use staining buffer + 0.1% of the permeabilization reagent (i.e. 0.1% Tween-20 or 0.1% Saponin).
3. Following the 15 minute incubation, add 2 mL of the staining buffer + 0.1% permeabilizer to each sample.
4. Centrifuge for 5 minutes at 400 RCF.
5. Discard supernatant and re-suspend in 1 mL of staining buffer + 0.1% permeabilizer.
6. Stain each sample at 1 uL/ 1 x 106 cells of primary antibody or 1-3 uL/ 1 x 106 cells for directly conjugated antibodies. Mix well and incubate at room temperature for 30 minutes- 1 hour. Gently mix samples every 10-15 minutes.
7. Following the primary/conjugate incubation, add 2 mL/sample of staining buffer +0.1% permeabilizer and centrifuge for 5 minutes at 400 RCF.
8. Remove supernatant and re-suspend each sample in 2 mL staining buffer + 0.1% permeabilizer, repeat wash for 5 minutes at 400 RCF.
9. If using a directly conjugated antibody, after the second wash, re-suspend cell pellet to a final volume of 500 uL per sample and proceed with flow analysis.

Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and wash the cells briefly in PBS. Add 10% formalin to the dish and fix at room temperature for 10 minutes.
2. Remove the formalin and wash the cells in PBS.
3. Permeablize the cells with 0.1% Triton X100 or other suitable detergent for 10 min.
4. Remove the permeablization buffer and wash three times for 10 minutes each in PBS. Be sure to not let the specimen dry out.
5. To block nonspecific antibody binding, incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
6. Add primary antibody at appropriate dilution and incubate overnight at 4C.
7. Remove primary antibody and replace with PBS. Wash three times for 10 minutes each.
8. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
9. Remove secondary antibody and replace with PBS. Wash three times for 10 minutes each.
10. Counter stain DNA with DAPi if required.

Immunohistochemistry-Paraffin Embedded Sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes (keep slides in the sodium citrate buffer at all times).

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in PBS for 5 minutes.
3. Block each section with 100-400 ul blocking solution (1% BSA in PBS) for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4 C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul HRP polymer conjugated secondary antibody. Incubate 30 minutes at room temperature.
7. Wash sections three times in wash buffer for 5 minutes each.
8. Add 100-400 ul DAB substrate to each section and monitor staining closely.
9. As soon as the sections develop, immerse slides in deionized water.
10. Counterstain sections in hematoxylin.
11. Wash sections in deionized water two times for 5 minutes each.
12. Dehydrate sections.
13. Mount coverslips.

Protocol for immunoprecipitation of LRRK2 followed by LRRK2 autophosphorylation kinase assay

Cell lysis
3X15 cm plates of SH-SY5Y cells were grown to 80% confluency. The plates were washed twice with PBS and placed on ice. Remaining PBS was aspirated off after tilting plate to remove all PBS. 1.5 ml of cold lysis buffer (buffer A) was added to each plate. The plates were allowed to incubate on ice 5 min until the cells detached. The lysis buffer and cells for each plate were then vigorously passed 6X through a 30.5 guage needle. Lysis buffer and cells were transferred to 3X1.5 ml eppendorf tubes and spun 5 min. at 5,000 rpm in a 4 degree eppendorf microfuge. Lysates were removed from pelleted debris and transferred to new 1.5 ml eppendorf tubes and recentrifuged 10 min. at 13,000 rpm. Lysate was transferred to three new tubes and 1/2 lysate volume of buffer A (-) NaCl was added to each tube.
Preclear
10 ug of rabbit IgG were added to the lysate for each tube and the lysate was vortexed followed by rotating at 4 degrees for 2 hours. 20 ul of protein A sepharose beads (Amersham cat#: 17-0469-01) were added. The tubes were vortexed and then rotated for 1.5 hours at 4 degrees. Lysates were separated from protein A beads beads by low (200 rpm) spin for 2 min and transferred to new eppendorf tubes. A repeat of the protein A sepharose incubation was carried out to remove residual rabbit IgG followed by removal of the protein A beads.

Immunoprecipitation with LRRK2 Ab

To two of the tubes containing precleared lysate were added 7 ul of LRRK2 Ab (7ug). To the remaining tube was added 7ug of rabbit IgG. The tubes were vortexed and allowed to rotate overnight at 4 degrees. The following morning 30 ul of protein A sepharose was added to each tube, the tubes were vortexed and rotated at 4 degrees for 2 hours. The protein A beads were then isolated by brief, low speed centrifugation and were washed 3X in 500ul buffer A (-) NaCl. This was followed by two washes in kinase buffer (buffer B). Protein A beads were resuspended in 1 volume (30 ul) of buffer B for a total of 60ul of immunoprecipitate.
Autophosphorylation kinase reaction, gel electrophoresis and phosphoimaging
On ice, 40 ul of immunoprecipitate from each tube was transferred to a.5ml kinase reaction tube. Each of the three reactions was supplemented with a 5 ul mixture that gave a final reaction concentration of 15 uM cold ATP and 5uCi ATP. The reaction mixtures were vortexed and transferred to a rotator in a 30 degree incubator. The autophosphorylation incubation was allowed to go for 30 minutes and the reaction tubes were taken off the rotator and vortexed every five minutes. The reactions were then halted by addition of 11ul of 5X SDS gel running sample buffer to each of the three samples. 40ul of each sample was then run on a 7% acrylamide-acetate mini-gel. Once the 200Kd molecular weight marker band had run half way down the gel, the gel was stopped dried and exposed blanked to a phosphoimaging cassette (Molecular Dynamics). Following 24 hour exposure, the cassette was assessed for radioactivity on a Storm analyzer.

Buffers

Buffer A (make 10ml both - and + NaCl solutions) = lysis buffer
50mM Tris pH 7.4
150mM NaCl
0.2% NP40
Protease inhibitor cocktail (stock = 100X, Sigma)
0.5mM vanadate
15mM EDTA
adjust to 10 ml with H2O
Buffer B = kinase buffer
10mM hepes
10mM MgCl2
50mM NaCl
protease inhibitor
vanadate
(1mM NaN3 if storing overnight or longer)

Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturer's instructions.