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WesternBlot_CarolinaSuplement

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					Biosci230: Protein Lab Con’t
Fish Muscle Protein Fingerprinting on SDS-PAGE & Actin Detection using Western Blotting

Lab Preparation (1st lab):

1.     Set up & put together SDS-PAGE gel unit with 1X running buffer.

2.     Receive from your instructors 8 samples (already boiled/3min):

       #1      10ul Protein Size Marker
       #2      10ul Shark sample
       #3      10ul Catfish sample
       #4      10ul Salmon sample
       #5      10ul Swordfish sample
       #6      10ul Tuna sample
       #7      10ul Flounder sample
       #8      10ul Orange Roughy sample

3.     Load ALL( 10ul) into each well, connect the electrodes, and run gel at 130 Volts for about
       1-1.5 hours.

4.     Remove the gel, trim off excess gel surroundings, place gel in distilled water for 5 min, discard
       d. water, & stain gel in Gel Code Blue dye buffer for at least 6 hours at room temp’t.

       NOTE: We will select 2 gels for Western Blotting, the rest will be stained in Gel Code Blue dye.
       After Western transfer using iBlot unit, the membranes will be stored in block buffer, and
       kept at 4C until next lab period.

       During 2nd lab period, we will use primary antibody against ACTIN cytoskeletal proteins & a Secondary
       antibody conjugated to Horse Radish Peroxidase (or Goat anti Rabbit-HRP) to visualize ACTIN cytoskeletal
       protein patterns in these different fish muscle samples.

5.     After 6 hours, discard gel staining solution, wash gel in d. water, & destain gel in d. water overnight.


Lab Preparation (2nd lab):

1.     From the 1st lab: transfer proteins on SDS-PAGE gel onto immobilized nitrocellulose (or
       synthetic PVDF) membrane as shown in video for iBlot unit:
       http://www.youtube.com/watch?v=WXvwe1n3ZYY

       Take out the membrane, cut off excess surroundings, block the membrane in blocking buffer, and
       incubate/store membrane at 4C until ready to probe with specific primary antibody.
2.    Prepare primary antibody [polyclonal actin antibody made in Rabbit species] at 1:1000
      dilution in 1X Phosphate Buffer Saline (pH7.4) – your instructor will prepare this solution:
      1 small W. Blot:
      10ml 1XPBS + 10ul of Polyclonal ACTIN primary antibody  1:1000 dilution

      Remove block buffer, add the primary antibody to the blot, & incubate blot at RT for 1 hour on a
      shaker unit.

3.    After 1 hour, discard the primary antibody, wash the blot with 20ml of 1XPBS/0.1%Tween-20
      detergent solution (wash buffer) at RT on a shaker unit for 10 min. Discard the wash buffer.

4.    Repeat step #3 TWO more times.

5.    Prepare secondary antibody [Goat anti Rabbit –HRP] at 1:2500 dilution in 1X Phosphate Buffer Saline
      (pH7.4) – your instructor will prepare this solution:
      1 small W. Blot:
      10ml 1XPBS + 4ul of Goat anti Rabbit secondary antibody  1:2500 dilution

      Discard the last wash buffer from step #4, add the secondary antibody to the blot, & incubate blot
      at RT for 1 hour on a shaker unit.
6.    After 1 hour, discard the secondary antibody, wash the blot with 20ml of 1XPBS/0.1%Tween-20
      detergent solution (wash buffer) at RT on a shaker unit for 10 min. Discard the wash buffer.

7.    Repeat step #6 TWO more times.

8.    Developing step:
      Your instructor will prepare FRESHLY made NBT/BCIPT substrate solution (~5ml/blot)
      Substrates will be added onto the blot, bands will appear within 5-10 minutes or so, record down the
      band intensity and the time it took to see the bands on the blot.
      Stop the developing process by washing blot with 1X PBS, take a picture of your blot and compare it
      to the gel that was stained with Gel Code Blue from the last lab period.

9.    Answer questions from the hand-outs.

References:
Monoclonal & Polyclonal Antibody Production:

http://www.youtube.com/watch?v=7ymKofaHCoY

&

http://www-users.med.cornell.edu/~jawagne/Antibody_Approaches.html

				
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