Detecting Expression of CAS9

Gain Trust That Your Gene Editing is on Target CRISPR/Cas9 is a hot genome-editing technology. However, the high frequency of off-target activity is a major issue.
One way to make sure CAS9 is being properly expressed is using markers against related proteins.

We are please to introduce our CAS9 markers. We plan on adding to these in the near future.

Image: Transfected Hek293 cells overexpressing the N-terminal 1-608 amino acids of S. pyogenes Cas9 . The cells were stained with MO22174 in red, and these cells also appear yellow since we counterstained with our rabbit Cas9 antibody, RA22126 to the same construct in green, giving a yellow color. The N-terminal construct contains a nuclear localization sequence and so is predominantly nuclear in localization. Most Hek293 cells in this field are not transfected so only the nuclei of these cells can be visualized with the blue DAPI DNA stain.

Name	Catalog #	Type	Applications
Cas9 from Steptococcus pyogenese	MO22174	Mouse IgG	ICC; IF; WB
Cas9 from S. pyogenese	RA22126	Rabbit IgG	ICC; IF; WB
Cas9 from Staphylococcus aureus	MO22175	Mouse IgG	ICC; IF; IHC; WB

We will post new CAS9 marker additions here.

Protein Purification Solutions

Strep-tag®Tools + Strep-Tactin Columns

We are seeing a notable uptick in use of our Protein Purification Tools. This includes vectors and purification columns. We are especially delighted with the increased use of of our Strep-Tag + Strep-Tactin Columns.

Figure: Strep-Tag+Strep-Tactin Methods

Here's an example of researchers using the technology to purify heme proteins for the study of the diptheria pathogen: Courtni E. Allen and Michael P. Schmitt. Utilization of Host Iron Sources by Corynebacterium diphtheriae: Multiple Hemoglobin-Binding Proteins Are Essential for the Use of Iron from the Hemoglobin-Haptoglobin Complex. J. Bacteriol. February 2015 vol. 197 no. 3 553-562. doi: 10.1128/JB.02413-14...Cell debris was removed by centrifugation at 12,000 × g at 4°C, and the supernatant fraction containing the soluble protein was purified on Strep-Tactin columns (Neuromics) for Strep-tagged constructs...

Figure: (A) HtaA and derivatives were assessed for the ability to bind Hb-Hp by ELISA. GST was included as a negative control. Experiments were repeated at least three times, with similar results. Results of a representative experiment are shown. (B) Binding of HtaA and various derivatives to Hb-Hp at a 200 nM concentration is shown. Binding of HtaA-Y361A and CR2-Y361A to Hb-Hp was significantly different from binding with HtaA-wt and CR2, respectively (**, P less than 0.0001). Binding of CR1 to Hb-Hp was significantly different from that with HtaA-wt (***, P less than 0.05). *, HtaA does not bind Hp (HtaA-Hp). Values represent the means from three independent experiments (±SD).

Looking for protein purification solutions? Do not hesitate to contact me: 612-801-1007 or pshuster@neuromics.com.

CRISPR-Cas9 and Sleeping Beauty Transposons

Neuromics-B-Mogen Approach

The CRISPR-Cas9 gene editing approach is now officially considered a revolution. This is confirmed by the increasing frequency of this word being used across the web in describing this technique. In fact, it is now appearing in conference titles. see: Genome Engineering: The CRISPR/Cas Revolution 2016COLD SPRING HARBOR - 17 AUG 2016.

We have joined the revolution with our new Sleeping Beauty Transposon^TM Systems.

Figure: Sleeping Beauty TransposonTM Systems Gene Integration Process. (A) A depiction of Sleeping Beauty transposon plasmid and Sleeping Beauty transposase enzyme active in cellular nuclei. (B) Transposase enzyme binds to Sleeping Beauty-specific IR/DR sites. (C) Transposase enzyme excises transposon sequence from transposon vector. (D) Transposase enzyme stably integrates transposon sequence at TA site in host cell genome.

Features Include:

• Simple-"Cut and Paste" Integration and Editing. Check out our Sleeping Beauty Transposon System Users Manual 
• Potent-100% Stable with No Off target effects with the safest transposon insertion profile. 
• Fast- Transfect with our transposase + Sleeping Beauty Transposon Reporter Series Vectors. 
• Cost Effective- Starting at $329.

If you would like us to engineer custom Transposons or gene editing on your cells, please conatact me directly and we will work together to formulate a Statement of Work to your specifications. Pete Shuster, CEO and Owner, Neuromics, pshuster@neuromics.com or cell phone: 612-801-1007