Western Blot protocol

Background:

The western blot experiment involves the usage of a nitrcellulose membrane after a SDS-PAGE gel electrophoreies. Gel electrophoresis is able to seperate a mixture of proteins by moleular wieght and then hae those result be tranfered onto the membrane to produce bands for the proteins. Once the anitbodies are added, those that havent bound to any proteins bands are washed off to allow those that are bound to be visibly seen. HPR solutions is what is added in order to visualize the protein bands and confirm or deny the potential identity of the unknown protein.

Purpose:

The purpose of this lab is to further determine the identity of unknown protein 3 which was though to be Myosin from mouse. The Experiments usage of western blot which uses anitbodies to accurately confirm or deny the suspected identity.

Hypothesis: If the correct antibody is added, the identity of the unknown protein will be discovered

Protocol

SDS-Page Gel Electrophoresis

  1. Obtain a small eppendorf tube and pipette 40uL of sample volume, 20uL of PBS, and 60uL of Sample Buffer.

  2. Boil in the thermocycler

  3. Next prepare the gel apparatus

  4. Obtain a gel and add buffer

  5. Once the gel apparatus is prepared, pipette 5uL of molecular weight marker in the first well. Pipette 15uL from the eppendorf tube into the next two gel wells. repeat patter up till well number 9.

  1. Run the gel for thirty mins at 200v .

  2. Next crack the gel open and prepare the transfer sandwhich.

  3. First open the cassete and black the black side on the bottom

  4. Layer a soaked sponge with the blocking buffer on the first layer,

  5. Next place a soaked filter paper

  6. Place the Gel on the filter paper

  7. Place the nitrcellulose membrane on top

  8. Place another filter paper on top and finally another sponge on the very top

  9. Close the transfer cassette

  10. Place the transfer cassette in the apparatus wiht the black side of the cassete facing the black and the ride facing the red.

  11. Fill with blotting buffer

  12. Place a ice pack and a magenetic stir rod inside with the cassette

  13. Run at 100V and turn on the stir plate

  14. Once done place the casette in a ziplock

Addition of the Antibodies

  1. Block the blot for thirty minutes

  2. Cut the blots into three sections and place each into different trays.

  3. Label the weigh boats with corresponding additions of anti-bodies

  4. add 10L of working buffer to all weigh boats

  5. The TA will add primary anti bodies

  6. Place on the rocker for 30 mins

  7. Pour out the solutions, add 10mL of working buffer to each weigh boats and place on rocker for five mins, repeat 3 times for an equivalent of three washes

  8. Pour of the solution of the third wash and add 10mL of working buffer to each weigh boats

  9. Add secondary anti body and place on rocker for 30 mins

  10. Pour the solutions and add 10mL of working buffer to each weigh boat and place on rocker for five mins. Repeat a total of three washes

  11. Pour out the third wash and rise with diH2O

  12. Add 10mL of HRP to each weigh boat and wrap with tin foil.

  13. Place on the rocker for 2-3 minutes until the bands show up

  14. Observe the data

Data/Dilutions Chart

Expected:

The results will reveal that the antibody band will appear next to the molecular weigh marker therefore will allow us to indicate what is the identity of the unknown protein.