OriGene now offers the service to generate Mass Spectrometry (MS) standards for 6,000 human proteins. Produced in human HEK293T cells and labeled with [U- 13C6, 15N4]-L-Arginine and [U- 13C6, 15N2]-L-Lysine, the full length proteins with appropriate post-translational modifications are the best identification and quantification standards.
Supriority of OriGene’s MS standards:
- Spiking at the early stage of sample process for accurate quantification
- Identify the best SRM and MRM transitions through experimental data
Internal standards of quantitative MS in cancer biology
- Authentic posttranslational modifications by using human HEK293T cell line.
- Higher data consistency than synthetic peptide internal standard
- Suitable for all types of MS equipments
- Over 90% incorporation efficiency.
1. Do a kill-curve to find out the lowest dose to kill your cells (4-7 days for puromycin). You can do that any time. You just need to know what concentration to use to select for your stable transfected clones.
Split your cells at 1:10 the day before the selection. Change the medium with different concentration of puromycine every 2 to 3 days. The normal conc. used for Puromycin is from 0.1 ug/ml to 10 ug/ml. So you can pick up a series of conc. within this range. For example, you can use 6 well plates, with each well containing a different conc. of puromycin (0, 0.1, 0.5, 1, 2, 10). You can pick more point if you want, so that you can get more accurate data. Check your cells every day, after 1 week, find the lowest dose that kill all your cells (100%). That’s the concentration you want to use for selection.
2. Perform a transfection with the plasmid containing Puro resistant gene. Twenty-four hrs post-transfection, passage the cells at 1:10 into fresh growth medium containing puromycin at the concentration you found from step 1. A mock transfection should be performed in parallel as a control. Grow and passage the cells as necessary (usually 2-3 days), maintaining selection pressure by keeping puromycin in the growth medium. After 1-2 weeks, a large number of the cells will be killed; the cells that remain growing in the selective medium have retained the expression plasmid, which stably integrates into the genome of the targeted cells. Monitor the mock control to ensure the cells are dying, no cells attached at the bottom.
3. If you pool all the colonies, that is your stable pool. If you isolate individual colony, that is your individual clones. For isolation of individual colony, there are several commercial tools to do that. You can also keep on diluting your stable transfected cells to the point that you can assume every well only get one colony. Continue growing these cells in selection medium for 1-2 additional passages. At this time, each well contains a clonal population of stably transfected cells, which can be maintained in normal growth medium without the selection pressure of puromycin (although you may wish to grow the cells under “light pressure”, lower concentration of puromycin). These populations can be used for experiments or stored under liquid nitrogen in growth medium with 10% DMSO and 20% FBS for future use.
Western blot analysis
- Run SDS-PAGE gel, and then Western transfer the protein samples to nitrocellulose (NC) membrane for immunoblot analysis.
- After transfer, transfer the membrane to western-blot tray, briefly wash the NC membrane with distilled water.
- (Optional) Visualize the proteins on the membrane by Ponceau’s staining.
- Wash off the red stain with distilled water.
- Block the membrane with 5-10ml blocking buffer (made by 5% non-fat milk in 1xPBST) for 30 minutes at R/T.
- Dilute the primary antibody with blocking buffer according to the suggested dilution factor on datasheet (In case of anti-DDK mouse monoclonal antibody (TA100011, do 1:4000 dilution).
- Remove the blocking buffer and add enough diluted primary antibody to cover the membrane.
- Incubate the membrane with primary antibody for 1hr at R/T. (Note: Or you can do overnight incubation at 4C, make sure you cover the western-blot tray to prevent excessive evaporation). To prevent uneven coverage, the western-blot tray can be rocked on a rocker platform.
- Collect the primary antibody and store them at 4C for up to two weeks. (If you would like to store them longer, you can freeze the diluted antibody at –20C. Remember frequent freezing and thawing will gradually decrease the antibody titer.)
- Briefly wash the membrane with 1xPBST once to remove any excessive primary antibody.
- Add enough 1xPBST to cover the membrane and leave the Western-blotting tray on a rocker platform.
- Wash the membrane for 15 minutes. (Note: If the background is high, repeat this step for two to three times.), turn on the developer during the wash time.
- Dilute HRP-conjugated secondary antibody with blocking buffer (1:5000 or higher dilution is usually good for Goat anti-mouse-HRP; TA100015).
- Incubate the membrane with secondary antibody for 30 minutes to 1hr.
- Wash the membrane with 1xPBST for 15 minutes, and then 3 times (5 min/time).
- Prepare the chemiluminescence development substrate mixture by mixing equal amount of solution 1 and 2 (TA100016; Normally 1ml will be enough for one membrane).
- Prepare a plastic saran film, lay the film on a flat surface, and dispense 1ml of substrate mixture for one membrane on the plastic saran film.
- Use a forceps to take washed the blot from the western-blotting tray, flip it, lay on the substrate mixture, and then incubate for 1 to 5 minutes. (Note: To avoid air bubbles, always lay the blot by touching one edge first.)
- Remove excess Chemiluminescence Reagent and wrap the membrane in plastic. Place inside X-ray cassette.
- Expose to film and develop
1xPBS: This buffer is made by dissolving 8g of NaCl, 0.2g of KCl, 1.44g of Na2HPO4 and 0.24g of KH2PO4 into 800ml of distilled water. Then adjust the pH to 7.4 with HCl, and add H2O to 1 liter.
1xPBST: 0.05% Tween 20 in 1xPBS
There are four sites in pCMV6-Entry than can be used to move the insert of a TrueORF clone into any of Gateway’s entry vectors (pENTR-1A, -2B, -3C, -4, and -11). These sites are BamH I and Kpn I (5′ end) and Not I and Xho I (3′ end). BamH I, Kpn I, and Not I will preserve the frame of the ORF; Xho I will not, and should only be used to transfer clones that will not be tagged at the 3’ end.
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.