Yu:Western: Difference between revisions
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===Transfer=== | ===Transfer=== | ||
{| border="0" cellpadding="10" | |||
|width="800px" valign="top"|1.Make 1L 0.5x TRIS-Glycine [[Yu:Western_Transfer_Buffer|Western Transfer Buffer]] from 10x stock and assemble the western transfer tank. | |||
|- | |||
|2. Carefully assemble the transfer sandwich, rolling to remove air bubbles at each step. | |||
::'''Black (Negative) Side''' | |||
:#Sponge | |||
:#2 pieces of Whatman chromatography paper | |||
:#SDS-PAGE Gel | |||
:#Nitrocellulose membrane | |||
:#2 pieces of Whatman chromatography paper | |||
:#Sponge | |||
::'''Red (Positive) Side''' | |||
|- | |||
|3. Transfer at room temperature for 1 hour at constant 100 volts. Current should range from 200-350 mA. | |||
|- | |||
|4. Remove nitrocellulose membrane and Ponceau stain in a Western blotting tray. Mark any protein ladders with a dull #2 pencil. | |||
|} | |||
===Immunoblotting=== | ===Immunoblotting=== | ||
{| border="0" cellpadding="10" | |||
=== | |width="800px" valign="top"|1. Make blocking buffer for Western blot and stir until fully dissolved. Store at 4°C | ||
:{| border="1" cellpadding="10" cellspacing="0" | |||
|width="300px" valign="top"|'''Solution''' | |||
|width="100px" valign="top"|'''Quantity''' | |||
|- | |||
|Carnation instant milk | |||
|5g | |||
|- | |||
|[[Yu:PBS#PBST||PBST]] | |||
|100mL | |||
|} | |||
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|2. Block nitrocellulose membrane on rocker for 20-30 minutes at room temperature with blocking buffer, or overnight at 4°C. | |||
|- | |||
|3. Discard blocking buffer and add primary antibody. Incubate on rocker at room temperature for 1 hour, or overnight at 4°C. | |||
|- | |||
|4. Draw off and save primary antibody for reuse. Rinse with 30-50mL PBST. | |||
|- | |||
|5. Wash twice on rocker for 10 minutes in PBST. | |||
|- | |||
|6. Make secondary antibody (2μL into 10mL Western Blocking Buffer) and incubate membrane on rocker at room temperature for 1 hour. | |||
|- | |||
|7. Discard secondary antibody. Rinse with 30-50mL PBST | |||
|- | |||
|8. Wash twice on rocker for 10 minutes in PBST. | |||
|- | |||
|9. Wash with distilled H<sub>2</sub>O for 10 minutes. | |||
|- | |||
|10. Develop and image. | |||
|} | |||
|} | |||
===Stripping=== | ===Stripping=== |
Revision as of 10:27, 2 December 2009
Western Blotting
SDS PAGE
1.Cast SDS-PAGE gel and assemble the gel apparatus. |
2. Add 10μL of desired sample and 5μL 3x Gel loading buffer to an eppendorf tube and boil for 5 minutes. Do this in separate tubes for each sample to be analyzed. After boiling, samples may be kept at room temperature until loaded |
3. Load 12μL to 15μL of sample into each lane. Load protein prestain marker, as well as BenchMark, if desired. Load any empty lanes with 1x Gel loading buffer |
4. Run gel until the loading buffer is just running off the bottom of the gel |
Transfer
1.Make 1L 0.5x TRIS-Glycine Western Transfer Buffer from 10x stock and assemble the western transfer tank. |
2. Carefully assemble the transfer sandwich, rolling to remove air bubbles at each step.
|
3. Transfer at room temperature for 1 hour at constant 100 volts. Current should range from 200-350 mA. |
4. Remove nitrocellulose membrane and Ponceau stain in a Western blotting tray. Mark any protein ladders with a dull #2 pencil. |
Immunoblotting
1. Make blocking buffer for Western blot and stir until fully dissolved. Store at 4°C
| |||||||
2. Block nitrocellulose membrane on rocker for 20-30 minutes at room temperature with blocking buffer, or overnight at 4°C. | |||||||
3. Discard blocking buffer and add primary antibody. Incubate on rocker at room temperature for 1 hour, or overnight at 4°C. | |||||||
4. Draw off and save primary antibody for reuse. Rinse with 30-50mL PBST. | |||||||
5. Wash twice on rocker for 10 minutes in PBST. | |||||||
6. Make secondary antibody (2μL into 10mL Western Blocking Buffer) and incubate membrane on rocker at room temperature for 1 hour. | |||||||
7. Discard secondary antibody. Rinse with 30-50mL PBST | |||||||
8. Wash twice on rocker for 10 minutes in PBST. | |||||||
9. Wash with distilled H2O for 10 minutes. | |||||||
10. Develop and image. |
|}
Stripping
Stripping Buffer
Solution | Volume |
1.5M Tris pH 6.8 | 2.08 mL |
β-Mercaptoethanol | 0.5 mL |
10% SDS | 10 mL |
milliQ H2O | 37.42 mL |
Final Volume is 50 mL |
Procedure