Protocols > Simultaneous Western blot

Simultaneous Western blot using three different primary antibodies on the same blot.


simulatenous western blot detection of RbcL, PsbA and PsaC in various species
1 μg of a total cell extract from (1) Arabidopsis thaliana, (2) Zea mays, (3) Anabaena sp., (4) Chlamydomonas reinhardtii, and (5) Thalassiosira pseudonana were separated on the Invitrogen NuPage system (Bis-Tris gel) for 50 min in MES buffer, then transferred to the Immobilon low FL 0.45 µm pvdf for 50 min.  M - Biorad "WesternC" marker which did not cross-react with antibodies was used. Protein standard mix (7-12): RbcL (AS01 017S), PsbA (AS01 016S), PsaC (AS04 042S) in serial dilution 6X with a starting volume of 800 fmoles.

Primary antibodies: rabbit anti-RbcL (AS03 037), chicken anti-PsbA (AS01 016) and rabbit anti-PsaC (AS10 939) were applied in 1: 2000 dilution followed by a standard washing protocol.  The following secondary antibodies were used:  goat anti-chicken DyLight® 488 conjugated (AS09 622) in dilution 1: 2000 (to detect PsbA) and goat anti-rabbit DyLight® 549 conjugated in dilution 1: 1000 (to detect RbcL and PsaC) followed by a standard washing protocol. 

The membrane was exposed for 3 s under the multiplex setting (DyLight ® 488 and DyLight ® 549) on the Biorad VersaDoc 4000 MP machine.  Details on the excitation sources and emission filters of the VersaDoc can be found here.

Note: primary antibodies to use in such a set up should be of equal titer.

Chosen Agrisera secondary antibodies

Anti-chicken DyLight®-conjugated secondary antibodies

AS11 1820 DyLight® 350 (violet)
AS16 3264 DyLight® 405 (blue)
AS09 622 DyLight® 488 (green)
AS11 1822 DyLight® 550 (yellow)
AS11 1823 DyLight® 594 (orange)
AS11 1824 DyLight® 633(red)
AS11 1825 DyLight® 650 (red-brown)
AS11 1827 DyLight® 800 (near-IR)

Anti-rabbit DyLight®-conjugated secondary antibodies

AS11 1812 DyLight® 350 (violet)
AS16 3510 DyLight® 405 (blue)
AS16 3527 DyLight® 488 (green)
AS11 1814 DyLight® 550 (yellow)
AS11 1815 DyLight® 594 (orange)
AS11 1816 DyLight® 633 (red)
AS12 2331 DyLight® 650 (red-brown)
AS16 3517 DyLight® 680 (far-red)
AS12 2456 DyLight® 800 (near-IR)

DyLight® is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.