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1.
Phospho-2D Western Blot
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Example 1.
Jurkat cells were treated with serum starvation for 1 hour
followed by adding serum to the culture medium, and culture
for 4 hours before harvesting the cells. Protein extraction
was prepared using 2D lysis buffer using Applied Biomics
protocols. First, sample was run on 2D DIGE (pH4-7)
and the gel image was scanned using Typhoon 9400 (A).
Second, proteins from the 2D gel were transferred
to the membrane and Western Blot was performed using mouse
monoclonal anti-phosphotyrosine antibody (Clone PY20) and
CF488-labeled goat anti-mouse Ig (Biotium, Hayward, California).
The Western Blot image was scanned using Typhoon 9400 (B)
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A. Gel image captured before the transfer
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B. Fluorescent 2D Western Blot using
anti-pTyr
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Example 2.
Hela cells were treated with 1 mM sodium orthovanadate for 30
minutes, and then protein extraction was prepared using 2D lysis
buffer using Applied Biomics protocols. First, sample
was run on 2D DIGE (pH4-7) and the gel image was scanned using
Typhoon 9400 (C). Second, proteins from the 2D
gel were transferred to the membrane and the membrane image
is captured (E). Western Blot was performed using mouse
monoclonal anti-phosphoserine antibody (Sigma) and CF488-labeled
goat anti-mouse Ig (Biotium, Hayward, California). The Western
Blot image was scanned using Typhoon 9400 (D and F).
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C. Gel image captured before the transfer
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D. Fluorescent 2D Western Blot using
anti-pSer
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E. Protein image captured from membrane
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F. 2D Western blot using anti-pSer(black and white)
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Example 3.
Hela cells were treated with 1 mM sodium orthovanadate
for 30 minutes, and then protein extraction was prepared using
2D lysis buffer using Applied Biomics protocols. First,
sample was run on 2D DIGE (pH4-7) and the gel image was scanned
using Typhoon 9400 (G). Second, proteins from
the 2D gel were transferred to the membrane and Western Blot
was performed using pooled mouse monoclonal antibodies against
phosphotyrosine, phosphoserine and phosphothreonine. The Western
Blot image was scanned using Typhoon 9400 (H)
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G. Gel image captured before the transfer
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H. Western Blot: anti-pSer, anti-pTyr
and anti-pThr
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Example.
Proteins were extracted from ovarian epithelial cells or mouse
liver (treated after long-term feeding with 3,5-diethoxycarbonyl-1,4-dihydrocollidine
(DDC), using Applied Biomics 2D DIGE protocol.
Green color: Transferred protein;
Red color: Anti-Ubiquitin.
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A. Protein
– Gel image (mouse liver)
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B. Protein
– Gel image (ovarian cells)
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C. Anti-Ubiquitin Western Blot
(mouse liver)
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D. Anti-Ubiquitin Western Blot
(ovarian cells)
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E. Protein + WB
overlay (mouse liver)
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F. Protein + WB
Overlay (ovarian cells)
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Example:
Proteins were extracted from mouse liver (treated after long-term
feeding with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC),
or from rat liver, using Applied Biomics 2D DIGE protocol.
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A. Protein – Gel image (mouse
liver)
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B. Protein – Gel image (Rat
liver)
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C. Anti-Acetylated-Lysine WB
(mouse liver)
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D. Anti-Acetylated-Lysine WB
(Rat liver)
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E. Protein + WB
overlay (mouse liver)
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F. Protein + WB
Overlay (rat liver)
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Example:
Proteins were extracted from Mouse mammary epithelial cells,
using Applied Biomics 2D DIGE protocol. |
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A. Protein
– Gel image (mouse cells)
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B. Anti-Sulfo-Tyrosine
WB
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C. Protein + WB
overlay
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Example:
Proteins were extracted from Mouse mammary epithelial cells,
using Applied Biomics 2D DIGE protocol.
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A. Protein
– Gel image (mouse cells)
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B. Anti-Methyl-Arginine
WB
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C. Protein
+ WB overlay
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Example:
Proteins were extracted out from Mouse liver (treated after
long-term feeding with 3,5-diethoxycarbonyl-1,4-dihydrocollidine
(DDC), using Applied Biomics 2D DIGE protocols.
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A. Protein
– Gel image (mouse cells)
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B. Anti-Nitrotyrosine
WB
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C. Protein + WB
overlay
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Example
Proteins were extracted from human lung cancer cells. 2D DIGE
gel was run on using pH 3-10 linear IPG strip and then transferred
to the PVDF membrane.
A: Membrane 2D image was scanned after transfer
B: Protein biotinalytion (2) was revealed using CF647-labeled
strepavidin (Biotium, Hayward, California)
C: Image overlay using ImageQuant software
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A. Protein
– Membrane image
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B. Biotin blotting
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C. Protein + Biotin
blotting overlay
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Call our tech support at 510-887-0889
or send email to support@appliedbiomics.com
with any questions.
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Different Staining
of 2D Gels
