BCH 4033L- lab 9: western blotting

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12 Terms

1
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what were the components of the electrophoresis buffer?

25 mM

192 mM

0.1% SDS

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what were the components of the transfer buffer?

25 mM tris

192 mM glycine

20% methanol

3
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what does SDS-PAGE stand for?

sodium dodecyl sulfate polyarcylamide gel electrophoresis

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how does SDS-PAGE work?

SDS binds and confers negative charge to proteins

SDS uniformly binds proteins

5
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what happens when an electrical field is applied to a PAG?

the neg. charged proteins are moved through the polyacrylamide matrix toward the anode

migration rate depends on size of proteins

6
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what are the membrane types used in western blotting applications?

polyvinylidene fluoride (PVDF) and nitrocellulose

can be done using wet or semi-dry transfer

7
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is wet or semi-dry faster?

semi-dry

requires less volume of reagents

produces comparable results with the wet transfer system

wet transfer less prone to failure due to drying of membrane for large proteins

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what stain is used?

ponceau-S red or copper phthalocyanine tetrasulfonic acid

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timings

wet membrane in ethanol for 15 seconds

decant methanol and soak membrane in dH2O for 2 minutes

equilibrate for 5 minutes

transfer at 15 V for 30 minutes, current limit of 5.5 mA/cm2

remove membrane and soak in 100% methanol for 10 seconds

immerse blot in 20% methanol for 5 minutes

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set up

cathode (negative)

buffer soaked filter paper (2)

nitrocellulose membrane

buffer soaked filter paper (2)

anode (positive)

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other techniques that use antibodies to erect proteins

immunofluorescence → seeing proteins in cells

enzyme-linked immunosorbent assay (ELISA) → IDing biological fluids

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how is ponceau staining done?

prior to immunostaining

submerge membrane with stain and put on rocker for 15 mins

remove membrane, rinse with dH2O

capture image

ensures that proteins have transferred from gel to membrane and blotting was successful