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Description: 

In this experiment, we want to want to compare the levels of endogenous cofilin and TEVp-Cofilin expressed under an inducible promoter with different levels of dox induction. This will allow us to determine what level of expression of cofilin would lead to the best signal:noise ratio of exogenous cofilin to endogenous cofilin. We are planning to transfect HEK293 with TEVp-Cofilin  under a TRE promoter and induce with varying levels of dox. We will then preform a Western blot with an anti-cofilin primary antibody and compare band sizes to test for the relative amounts of endogenous cofilin and exogenous TEVp-Cofilin  in the cells.

 

Cloning

COMPLETE

Description

Making pEXPR TRE:TEVp-cofilin.

Plan
  •  Order TEVp-cofilin 
  •  Golden Gate gBlocks into ggDONR
  •  Transform bacteria with Golden Gate product and plate 
  •  Pick colonies and grow in liquid culture
  •  Miniprep cells and verify product 
  •  Make glycerol stocks
  •  LR into pDEST
  •  Transform, pick colonies and grow in liquid culture
  •  Miniprep cells and verify product
  •  Make glycerol stocks 
  •  Midiprep product
Status
  • We have not yet received gBlock

    • Experiment

    Transfection Plan:

    Plate 1

    Well 1

    HEK293

    Well 2

    HEK293

    + Dummy (

    1,000ng

    2500ng)

    Well 3

     

    HEK293

    + Dummy (

    500ng

    1500ng)

    +hEF1a:

    GFP

    YFP (

    500ng

    1000ng)

    Well 4

     

    Well 5
    Well 6

     

     

      Image Added

     

     

    		  

     

    Plate 2
    Well 7

     

    HEK293

    +hEF1a:

    GFP

    YFP (

    400ng

    1000ng)

    +hEF1a:rtTA (

    200ng

    500ng)

    +TRE:TEVp-Cofilin (

    400ng

    1000ng)

    0x
    0nM Dox
    Well 8

     

    HEK293

    +hEF1a:

    GFP

    YFP (

    400ng

    1000ng)

    +hEF1a:rtTA (

    200ng

    500ng)

    +TRE:TEVp-Cofilin (

    400ng

    1000ng)

    1x
    1nM Dox
    Well 9

     

    HEK293

    +hEF1a:

    GFP

    YFP (

    400ng

    1000ng)

    +hEF1a:rtTA (

    200ng

    500ng)

    +TRE:TEVp-Cofilin (

    400ng

    1000ng)

    10x
    10nM Dox
    Well 10

     

    HEK293

    +hEF1a:

    GFP

    YFP (

    400ng

    1000ng)

    +hEF1a:rtTA (

    200ng

    500ng)

    +TRE:TEVp-Cofilin (

    400ng

    1000ng)

    100x
    100nM Dox
    Well 11

     

    HEK293

    +hEF1a:

    GFP

    YFP (

    400ng

    1000ng)

    +hEF1a:rtTA (

    200ng

    500ng)

    +TRE:TEVp-Cofilin (

    400ng

    1000ng)

    1,000x
    1000nM Dox
    Well 12

     

    HEK293

    +hEF1a:

    GFP

    YFP (

    400ng

    1000ng)

    +hEF1a:rtTA (

    200ng

    500ng)

    +TRE:TEVp-Cofilin (

    400ng

    1000ng)

    2,000x
    2000nM Dox
    Well 13
    Image Added
    Well 14
    Image Added
    Well 15
    Image Added
    Well 16
    Image Added
    Well 17
    Well 19
    Well 20
    Well 21
    Well 22
    Well 23
    Well 2
    Image Added
    Well 18
    Image Added

     

    Controls:

    GAPDH: Loading control (how many cells we've loaded onto the gel)

    YFP: Transfection marker (how many transfected cells we've loaded onto the gel)

    Protein Sizes:
    Protein
    Size (kDa)
    Cofilin18.5
    TEVp-Cofilin 48.6
    GAPDH37
    YFP27
    Gels:

    Image Modified

    We have the any kD gels in the lab (expires 2015) and this will work for both our western blot and antibody group's western blot.

    Image Modified

    If we run out of the above gel we are considering getting the 4-25% gel in order to optimize our resolution. This will also work for both our western blot and antibody group's western blot.

     

     

    Antibodies:
     
    Antibody
    Type
    Conjugation
    Dilution Used
    Link
    Anti-Cofilin/Cofilin-TEVpRabbit Monoclonal-

    T1:1/10000

    T2:1/2500

    		http://www.abcam.com/cofilin-antibody-ep6376-ab134963.html
    Anti-GAPDHMouse Monoclonal-1/10000http://www.abcam.com/gapdh-antibody-mabcam-9484-loading-control-hrp-ab9482.html
    Anti-eYFPRabbit Polyclonal-1/10000http://www.abcam.com/gfp-antibody-chip-grade-ab290.html
    Goat anti-mouse secondary IRDye
    680
    800-RD1/15000https://licor.secure.force.com/catalog/LI_ProductDetailsPage?sku=926-68070&store=bio&viewState=DetailView
    Goat anti-rabbit IRDye
    800
    680-CW1/15000https://licor.secure.force.com/catalog/LI_ProductDetailsPage?sku=827-08365&store=bio&viewState=DetailView


    Expected Results:

     LadderHEK293

    HEK293

    + Dummy

    HEK293

    +

    GFP

    YFP

    + Dummy

    HEK293

    +

    GFP

    YFP

    + TEVp-Cofilin 

    50    TEVp-Cofilin 
    40 GAPDH GAPDH GAPDHGAPDH 
    30  
     
    YFP
    GFP 
    YFP 
         
    20 Cofilin Cofilin
     
    Cofilin Cofilin
    Status: 
    • Not yet started - waiting on parts

     

     

     

     

    Results: 

    Expand
    titleTrial 1
    Lane1234567891011
    ContentsLadderUntransfectedDummyeYFP

    eYFP

    rtTa

    TEVp-Cofilin

    0nM Dox

    eYFP

    rtTa

    TEVp-Cofilin

    1nM Dox

    eYFP

    rtTa

    TEVp-Cofilin

    10nM Dox

    eYFP

    rtTa

    TEVp-Cofilin

    100nM Dox

    eYFP

    rtTa

    TEVp-Cofilin

    1000nM Dox

    eYFP

    rtTa

    TEVp-Cofilin

    2000nM Dox

    		Ladder
    Protein
    Size (kDa)
    Cofilin18.5
    TEVp-Cofilin 48.6
    GAPDH37
    YFP27
    		                            
    Image AddedImage AddedImage Added

    Odyssey blocked blot.

    Cofilin and TEVp-Cofilin bands are not there...

    We know that cofilin is present in relatively large amounts and the fact that neither native cofilin nor TEVp-cofilin bands appear might indicate that there is a problem with the anitbody or the antibody treatment of the blot. Both anti-cofilin and anti-eYFP are rabbit antibodies and since eYFP bands show, we can assume that the goat anti-rabbit secondary works.

    YFP and GAPDH bands are higher than they should be.

    Low signal from GAPDH (relative to YFP)

    BSA blocked blot.

    Cofilin and TEVp-Cofilin are not here either

    There's a lot of background

    		 
    Expand
    titleTrial 2

    Modifications to Trial 1:

    Increased cofilin primary antibody concentration from 1/10,000 to 1/2500 because both cofilin and TEVp-cofilin bands didn't show up in Trial 1

    --- Didn't help that much considering the cofilin bands are still not there

    Didn't probe for GAPDH because the amount of background that the 800 secondary antibody was producing

    --- No background (smile) (although, there wasn't that much background with the odyssey blocked blot in Trial 1)

    Also the YFP band (the only one there) is again slightly higher than it should be

    Lane1234567891011
    ContentsLadderUntransfectedDummyeYFP

    eYFP

    rtTa

    TEVp-Cofilin

    0nM Dox

    eYFP

    rtTa

    TEVp-Cofilin

    1nM Dox

    eYFP

    rtTa

    TEVp-Cofilin

    10nM Dox

    eYFP

    rtTa

    TEVp-Cofilin

    100nM Dox

    eYFP

    rtTa

    TEVp-Cofilin

    1000nM Dox

    eYFP

    rtTa

    TEVp-Cofilin

    2000nM Dox

    		Ladder
    Protein
    Size (kDa)
    Cofilin18.5
    TEVp-Cofilin 48.6
    GAPDH37
    YFP27

    Image Added    		Image Added

    Next step: I would like to have a positive control. Abcam suggests HeLa lysates (if we have those around in the lab)