It is very difficult to sequence the shRNA constructs due to the hairpin structure. However, you can use the DNA relaxation agents in the sequencing reaction (0.83 M Betaine, Sigma #B-0300), plus 1x PCRx Enhancer [in Invitrogen kit part # 11495-017]), which may be helpful.
Sequencing primers for OriGene Hush constructs:
forward primer is located at the 5′ of the cloning site (around ~188bp), and has the sequence 5` ACGATACAAGGCTGTTAGAGAG 3`.
reverse primer is ~70bp downstream of the last T of the 6T terminator. And the sequence is 5′-TTGAGATGCATGCTTTGCATAC-3′.
Are you sure that your IHC signals are truly specific?
High specificity is the pre-requisite for IHC antibody to be used for diagnostic and therapeutic applications. The cross-reactivity may potentially cause unexpected side effects and false diagnostic reports for clinicians. However the validation tools for antibody specificity are lacking. Although research data from various groups has shown that some monoclonal antibodies on the market are not mono-specific, it remains a challenge to produce the truly mono-specific antibodies to replace them.
OriGene is proud to announce that we have developed a high density protein microarray chip for antibody specificity testing. With the world’s largest collection of overexpression protein lysates, OriGene is in the unique position to produce a high density chip of human proteins. This protein chip is spotted with 10,464 (10K) unique over-expressed proteins in duplicate on a single nitrocellulose coated glass slide. This protein microarray technology has been used to validate the specificity of an existing ERCC1 diagnostic monoclonal antibody, and has been applied as a screening method to identify the most specific UltraMAB™ monoclonal antibody for this target.
The excision repair cross-complementation group 1 (ERCC1) protein is an important biomarker for clinicians to predict whether certain patient populations with non-small cell lung carcinoma will respond to cisplatin chemotherapy. As such, it is critical to develop highly-specific immunohistochemistry validated monoclonal antibodies for this diagnostic test. Several publications revealed that 8F1, the most commonly used antibody clone for ERCC1, exhibits cross-reactivity to an unknown protein in ERCC1 deficient cell lines. By using OriGene’s high-density protein microarray technology, the corresponding cross-reactive binding protein for 8F1 monoclonal antibody was identified. This technology also enable us successfully develop the most specific UltraMAB™ monoclonal antibody for ERCC1 (4F9). This data was further confirmed by western blot analysis.