Same antibody, different outcome on samples from wide taxonomic range

So-called "Garden blot" means that the same antibody is used on samples from wide taxonomic range.

In the presented example, samples were extracted from dicotyl plants, cyanobacteria, diatoms and algae. On the sequence level, the peptide which was used to elicit anti-RbcL antibody, AS03 037 is perfectly conserved in RbcL from all analyzed species. However, clear differences in RbcL band intensity can be observed.

This difference can be attributed to:

Differences in growth stages and conditions for analyzed samples
Differences in RbcL levels between analyzed species
Normalization done on chlorophyll is not appropriate for cyanobacteria which have phycobilisomes instead of chlorophyll based light harvesting

Even though the peptide or protein sequence which was used to elicit given antibody is perfectly conserved, there can be differences in intensity of visualized target protein band. For some protein-antibody pairs, 6-8 M urea needs to be included for successful detection, as described here.

Western blot detection of RbcL in a wide species range
0.25 µg of chlorophyl a/lane from Spinacia oleracea (1), Synechococcus PCC 7942 (2), Cyanophora paradoxa (3), Heterosigma akashiwo (4), Thalassiosira pseudonana (5), Euglena gracilis (6), Micromonas pusilla (7), Chlamydomonas reinhardtii (8), Porphyra sp (9), Gonyaulax polyedra (10), Emiliania huxleyi (11) extracted with PEB (AS08 300), were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to nitrocellulose. Filters were blocked 1h with 2% low-fat milk powder in TBS-T (0.1% TWEEN 20) and probed with anti-RbcL antibody (AS03 037, 1:50 000, 1h) and secondary anti-rabbit (1:20000, 1 h) antibody (HRP conjugated, recommended secondary antibody AS09 602) in TBS-T containing 2% low fat milk powder. Antibody incubations were followed by washings in TBS-T. All steps were performed at RT with agitation. Blots were developed for 5 min with ECL Advance detection reagent according to the manufacturer’s instructions (GE Healthcare). Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).

Same antibody, different outcome on samples from wide taxonomic range

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