WESTERN BLOT

**Western Blot**

~~ ULB +

Lysosomes have lots of proteases. We want to protect proteins as we lyse cells.

Lysosomes will not work when pH around 5.

  1. Protease Inhibitors 2. Phosphatase inhibitors

  • 1. Protease Inhibitors: Cocktail

    • Aptoninin: Serine protease inhibitor

    • Chymastatine: Serine and Cysteine protease inhibitor

    • Leupeptin: Serine and Cysteine protease inhibitor

    • Pepstain: general Inhibitor (blocks lots of Proteases)

  • 2. Phosphatase inhibitors: Cell signaling

    • Beta- glycerophotase *

    • Sodium molybdate *

    • Sodium othoranadate *

    • *main ones*

    • DMSF

  • 3. DTT : Breaks disulfide bridges

Make ULB (Lysis Buffer)

  • SDS

  • Tris

  • NaCl

  • NaF pH inhibitor

  • DTT

Do 5’ fold and 10’ fold dilutions with lysis buffer and do reading of those

  • whichever one gives you reading within the linear range of the assay is one you’ll use.

*Western Blot*

RNA —→ Protein

Translation —> Western Blot

  1. Isolate Protein from tissue or cells

  2. Lyse cells — make homogenate

Basis* ULB

Lysis Buffer (ULB):

  • 1. Detergent: (SDS) non-ionic detergent*

    • NP-40

    • Triton X100

    • 5% w/v

  • 2. Tris: pH regulator*

    • pH 7.5

    • 50 mM

  • 3. NaCl (salt) - 50 mM*

    • Dissolves into ionic form when mixed with H2O

      • Change the salinity of the solution around the cell

      • create hypotonic solution

      • lots of ions inside cells

      • Water rushes in, destabilizing the membrane

    • Lyses cell

  • 4. NaF: Phosphate inhibitor - 50mM

Make Buffer

  • Weight out solids

  • Add water (amt. based on solvent)

  • Add HCl to bring pH down to 7.5

  • Add water

Protein Assay:

Protein Assay: Bradford Assay

  • Commassile Brilliant blue G - 250 dye

    • Dye binds to basic amino acids arginine and aromatic a.a

      • changes to blue

    • More protein you have, more dye that binds, more blue you get

  • If we do lysis and have concentrated protein lysate we’ll have to serail dilute a couple of times.

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  • y=mx+b x is mult by 2 or 5.

  • look at notes for math portion

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SDS-Page: (gel electrophoresed for proteins)

  • Sodium dodecyl sulfate Polyacrylamide gel electrophoresis (8% -12%)

    • anionic detergent

  • Coats proteins

    • uniform negate charge

Acrylamide

  • thin, run up and down, separate proteins.

Glycine

  • an amino acid that can be protonated or deprotonated

TEMED

  • the catalyst causes the cross-pattern

APS

  • catalyst polymerizes acrylamide

Loading dye for SDS-PAGE

  1. Mix diluted proteins with loading dye

    1. Tris Buffer -7.5

    2. Bromophenol Blue

    3. Glycerol

    4. DTT

    5. Glycine

Gel components: what do they do?

Tris pH 6.8 standing

Tris ph 8.8 running

  1. Glycine

  2. SDS

  3. Acrylamide

  4. Bis - acrylamide

  5. APS

  6. TEMED

  7. H2O

Running buffer

  1. SDS

  2. Glycine

  3. Tris

Shape- linear

  • denature 95C for 5 min

  • Breaks disulfide bridges (DTT or Beta- mercaptoethanol)

    • Reducing agents

~~Transfer

  • Buffer

    • Glycine

    • Tris

Structure for gel stain

Metal plate

filter paper

PVDF membrane (methanol)

Gel

Filter paper

Metal plate

  • PVDF has the transfer

Stain membrane with

  • Ponceau S (red dye)

    • protein adhesión

  • Wash H2O

Block with milk in T-TBS

  • Clean off any weakly associated bonds

    • Saline weak ion and bonding

    • weak hydrophobic

Process:

  1. Milk - fill all open spaces on the membrane

  2. Wash T-TBS

    1. 3x -5min

  3. Incubate with 1` antibody diluted with MyHC

    1. in 5% milk + T-TBS (10mL)

    2. 1-2hr @RT

  4. Wash T-TBS

    1. 3x -5min

  5. Incubate with 2` antibody

    1. 5% milk + T-TBS (30-60 min)

  6. Wash T-TBS

    1. 3x -5min

  7. Develop

    1. light production

What is the purpose of using a secondary antibody for western blotting?

  • Bind to the primary antibody, Amplify the level of the detection signal, and Eliminate the need to enzyme-conjugate every primary antibody.