Apr 30, 2020

Public workspaceWestern Blot  V.1

DOI
dx.doi.org/10.17504/protocols.io.bfiijkce
  • Payal Patel1,
  • Bethany Rozeboom1,
  • Tarah-Anne Abrigo1
  • 1Mercer University, Biochemistry Senior Capstone
Bethany Rozeboom
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DOI: dx.doi.org/10.17504/protocols.io.bfiijkce
Protocol Citation: Payal Patel, Bethany Rozeboom, Tarah-Anne Abrigo 2020. Western Blot . protocols.io https://dx.doi.org/10.17504/protocols.io.bfiijkce
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 24, 2020
Last Modified: April 30, 2020
Protocol Integer ID: 36138
Materials
MATERIALS
Reagent2x Laemmli Sample BufferBio-Rad LaboratoriesCatalog #1610737
ReagentTE bufferThermo Fisher Scientific
ReagentBio Rad Precision Plus protein StandardBio-Rad LaboratoriesCatalog #161-0374
ReagentTryptic Soy Broth, E - 10mLThermo FisherCatalog #R07226
ReagentSignalFire™ ECL ReagentCell Signaling TechnologyCatalog ##6883S
ReagentTris Buffered Saline (TBS-10X)Cell Signaling TechnologyCatalog ##12498
ReagentTris-Glycine Transfer Buffer (10X)Cell Signaling TechnologyCatalog ##12539
ReagentTris-Glycine SDS Running Buffer (10X)Cell Signaling TechnologyCatalog ##4050
ReagentTris Buffered Saline with Tween® 20 (TBST-10X)Cell Signaling TechnologyCatalog ##9997
ReagentNonfat Dry MilkCell Signaling TechnologyCatalog ##9999
ReagentBSACell Signaling TechnologyCatalog ##9998
ReagentAnti-mouse IgG HRP-linked AntibodyCell Signaling TechnologyCatalog ##7076

Cell Preparation
Cell Preparation
Grow cell Amount50 mL of TSB, let incubate DurationOvernight

Distributed Amount17 mL of culture into two Amount50 mL centrifuge tubes, and Amount10 mL into one Amount20 mL centrifuge tube

Centrifuge down for Duration00:20:00 at 4300rpm ( 4303 xg) Temperature4 °C


Aspirate media from cultures; wash cells 3x with TE aspirate.
Lyse cells by resuspending in Amount1 mL of the different lysis buffers

Concentration20 millimolar (mM) Tris Cl , Concentration15 millimolar (mM) NaCl , 0.9696% Triton Ph7.5 (labelled A)
Phosphatase Inhibitor: Concentration50 millimolar (mM) NaF , Concentration1 millimolar (mM) Sodium Orthovanadate , Concentration10 millimolar (mM) Sodium Pyrophosphate and Concentration10 millimolar (mM) B-glycerophosphate
EDTA-free Protease tablet (1 tablet per Amount10 mL )

1% SDS, Amount10 mL Tris Cl , Concentration1 millimolar (mM) EDTA (labelled B)
Phosphatase Inhibitor: Concentration50 millimolar (mM) NaF , Concentration1 millimolar (mM) Sodium Orthovanadate , Concentration10 millimolar (mM) Sodium Pyrophosphate and Concentration10 millimolar (mM) B-glycerophosphate
EDTA-free Protease tablet (1 tablet per Amount10 mL )
Freeze thaw 3 times
Sonicated for Duration00:01:00 , repeat 5 times

Centrifuged at 13000 rpm for Duration00:05:00

Bradford Assay
Combine Amount40 µL 4X Laemmli to cell lysate, Amount40 µL of Lysis A and Lysis B

Load Amount40 µL o onto SDS-PAGE gel and Amount40 µL of All Blue Precision Plus for ladder


Soaked sponges and transfer paper in Transfer Buffer
Soak PVDF membrane in Methanol
Soak fiber pads thoroughly in a Transfer Buffer
Make the blotting sandwich
Add Thikness1 cm depth of Transfer Buffer to container and insert plastic cassette with black side down, lay a wet fiber pad on the black side of the cassette

Lay one wet blotting paper on the fiber pad and roll out air bubbles
Lay gel squarely on blotting paper and roll out air bubbles
Lay wet PVDF on the gel and roll out air bubbles
Lay one wet blotting paper on the membrane and roll out air bubbles
Lay a wet fiber pad on top of the blotting paper and close the cassette and clamp together with the white clip.
Electrotransfer to PVDF membrane at 30 mV DurationOvernight

Membrane Blocking
Membrane Blocking
After transfer, wash PVDF membrane with Amount25 mL TBS for Duration00:05:00 at TemperatureRoom temperature

Incubate membrane in Amount25 mL Blocking Buffer for Duration01:00:00 at TemperatureRoom temperature

Combine Amount7.5 g Nonfat dry Milk with Amount150 mL TBST to make Blocking Buffer

Wash three times forDuration00:05:00 each with Amount15 mL TBST
Incubate membrane in Primary Antibody (at 1:2000) in a Amount10 mL Primary Antibody Dilution Buffer with gentle agitation DurationOvernight
Combine Amount1.0 g 5% BSA to Amount20 mL 1X TBST and mix well

Wash three times for Duration00:05:00 each with Amount15 mL TBST

Incubate membrane with Anti-mouse IgG, HRP-linked Antibody (1:1000) to detect biotinylated protein markers in Amount10 mL Blocking Buffer with gentle agitation for Duration01:00:00 at TemperatureRoom temperature

Wash three times for Duration00:05:00 each with Amount15 mL TBST
Detection of Proteins
Detection of Proteins
Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (based off of the size of our membrane we mixed Amount1 mL of each together)

Incubate substrate with membrane for Duration00:07:00 , remove excess solution (membrane remains wet) expose to X-ray film using an imager