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titleProtocol

 

Lysis buffer (from Abcam)

pH 8.0

150 mM NaCl

10% NP40 or Triton X-100

50 mM Tris

 

Running buffer (from Bio-Rad)

pH 8.3

25 mM Tris

192 mM glycine

0.1% SDS

 

Gel stain (from Abcam)

0.3 M CuCl2

Gel destaining buffer (from Abcam)

pH 8.0

0.25 M Tris

0.25 M EDTA

 

Transfer buffer (from Abcam)

48 mM Tris

39 mM glycine

0.04% SDS

20% methanol

 

Membrane stain (Ponceau Red) - stock (from Abcam)

Dilute 1:10 for washing the membrane

2% Ponceau S

30% trichloroacetic acid

30% sulfosalicylic acid

 

TBST (Antibody/BSA buffer) (from Abcam)

100 mL TBS 10x (pH 7.6)

  • 24.23 g Trizma HCl
  • 80.06 g NaCl
  • to 1L with dH2O

900 mL dH2O

1 mL Tween20

 

Block in 5% BSA (5g BSA in 100 mL TBST)

 

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Data
titleResults
123456789101112
LadderUntransfectedDummy

hEF1a:eYFP

Dummy

hEF1a:eYFP

hEF1a:rtTA

TRE:Syk-TEVp

0 nM Dox

hEF1a:eYFP

hEF1a:rtTA

TRE:Syk-TEVp

1 nM Dox

hEF1a:eYFP

hEF1a:rtTA

TRE:Syk-TEVp

10 nM Dox

hEF1a:eYFP

hEF1a:rtTA

TRE:Syk-TEVp

100 nM Dox

hEF1a:eYFP

hEF1a:rtTA

TRE:Syk-TEVp

1000 nM Dox

hEF1a:eYFP

hEF1a:rtTA

TRE:Syk-TEVp

2000 nM Dox

 Ladder

 

 Image Added

 

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titleAnalysis
ProteinSize (kDa)
Syk72.08
Syk-(15aa)-TEVp100.5
GAPDH37
GFP27

Image Added

wrong ladder used - highest band = 40kd

gel not run long enough to distinguish larger proteins

loading control bands didn't show up - possibly because the blocking buffer we used contained casein (protein in milk) and abcam warned that using milk as a blocking buffer greatly reduced signal from anti-gapdh