Antibody specificity has been cited as a major cause of concern among users in independent literature (1-3) and is ranked as the most important factor for choosing a new antibody supplier. (4).
RNA interference (or RNAi) is the most definitive test of antibody specificity short of using knockdown animal models, yet has not been applied to the routine testing of antibodies in the antibody industry at present. Proteintech's RNAi procedure will be routine for all new products in the future and has already been retrospectively applied to existing products in several stages.
In rolling out its own RNAi testing, Proteintech will make a gold standard of antibody validation more accessible to all life scientists at a crucial stage: before they commit valuable time and resources to antibody-based experiments. This move sets a new industry benchmark, with perhaps the further-reaching implications of raising validation standards of commercial antibodies on the whole.
Proteintech siRNA validation showcase
siRNA AKT1 result from Dr. Eva Martinez-Balibrea. Green:tubulin. Red:10176-2-AP, AKT1
A549 cells (shcontrol and shRNA of TDP43) were subjected to SDS PAGE followed by Western blot with 10782-2-AP (TARDBP antibody) at dilution of 1:1000. (Data provided by Angran Biotech www.miRNAlab.com)
Proteintech antibodies included in the showcase:
Over 1000 antibodies in the Proteintech catalog have been tested with siRNA-treated samples.
|Antigen name||Cat No.||Application||Type|
|AKT antibody||10176-2-AP||ELISA,WB,IHC,IP,FC||Rabbit poly|
|TDP-43 antibody||10782-2-AP||ELISA,IF,WB,IHC,IP,FC||Rabbit poly|
Look out for icon on our datasheets. This icon shows when our antibodies have been tested in siRNA-treated samples.
Target site selection
Proteintech scientists obtain the target site from the available literature and online shRNA design tools.
Construction of pGenesil-1 shRNA expression vector
Proteintech scientists design two single-stranded 19-22mer DNA oligonucleotides: one encoding the target shRNA (sense strand) and the other encoding its complementary strand (antisense strand). These will eventually anneal together to generate a double-stranded oligonucleotide when expressed in the target cell.
The two loop sequences (sense and antisense) are linked together on the same strand by a short linker sequence that will form the hairpin loop structure upon expression. A variety of loop sequences have been successfully used by researchers, but Proteintech uses the loop sequence TTCAAGACG.
At the end of the shRNA template is a 6 nucleotide poly (T) tail recognized as a termination signal. The 5' ends of the 2 oligonucleotides are non-complementary and form the BamHI and HindIII restriction site overhangs that facilitate efficient directional cloning into pGenesil-1 vector.
RNAi is a relatively new technology employed for sequence-specific gene silencing in the lab. It is based on a biological process of the same name first discovered in plants and, later, the animal kingdom. In nature, RNAi has important roles in defending cells against parasitic nucleotide sequences (eg., from viruses and transposons) and it also influences development.
Its basic premise is as a means of gene expression control at the RNA level- the intermediate stage between gene transcription and protein translation- and works by preventing messenger RNA (mRNA) from being translated into protein. It is achieved experimentally in the lab by delivery of synthetic dsRNA or a plasmid DNA vector containing sequence coding for a small hairpin RNA (shRNA). The dsRNA or shRNA is then processed into single-stranded siRNA and this targets specific mRNA by complementary sequence pairing. Once target mRNA has been 'silenced' by siRNA binding, it is degraded via cellular processes. The RNAi method is a form of gene 'knockdown' and when successful it significantly reduces the levels of target protein below the threshold of Western blot detection. Specific antibodies can then be identified by an absence of (or significantly reduced) signal in shRNA-treated samples on a Western blot membrane.
Figure 1: An overview of how RNAi works, using the plasmid expression method to introduce the siRNA to cells.