Over-expression Cell Lysate FAQs
1. What are the terms of the OriGene over-expression cell lysate guarantee?
We promise guaranteed product quality and timely expert customer service. Our over-expression cell lysates are guaranteed for 12 months from the date of shipment if stored at conditions prescribed in the product insert. Each over expression lysate is validated by Western blot and the image showing the positive band at or near the correct molecular weight is provided.
2. How long can I store these lysates?
OriGene guarantees 12 month stability from date of shipment when these samples are stored at -20°C. Avoid repeated freeze-thaw cycles.
3. Does your SDS buffer already containing reducing reagents?
Yes, the SDS Sample Buffer contains 100mM DTT.
1 vial of 250ul 2xSDS Sample Buffer (4% SDS, 125mM Tris-HCl pH6.8, 10% Glycerol, 0.002% Bromophenol blue, 100mM DTT)
4. Do all TrueORF clones produce same amount of over-expressed proteins?
Each clone behaves differently as it depends on the protein that is expressed in the cell line. Each vial of over-expression cell lysates contains 100 ug of total protein and presence of the target protein is verified by Western blot (picture included in the product datasheet).
5. Can I use these over-expression cell lysates for other applications beside Western blots?
The over-expression lysates have been validated for western-blot analysis. Optimal dilution/concentrations should be determined by the end user.
6. How many Western blots can one do with 100 ug total protein in a lysate?
Each vial of cell lysate contains 100ug of total protein which should be sufficient for 20~50 reactions. Depending on over-expression level, antibody affinity and detection system, some lysates can go as low as 0.1-ug per load.
7. What should I do if I can't find specific lysate of my interest?
We routinely add new lysate products on a quarterly basis. Please check our website frequently or contact us to help us prioritize new product release schedule. OriGene will contact you as soon as the lysate product you requested is available.
8. Does OriGene sell Western blot MW standard?
Yes. We have a MYC/DDK Tagged Western Blot Molecular Weight Markers (MWM1001) for Western Blot applications.
9. What source do you use for the predicted molecular weight of the antigen?
We mainly use the Swiss-Prot database to predict the molecular weight of an unprocessed protein. For details of the database, visit the ExPASy Bioinformatics Resource Portal.
10. If the western-blot analysis shows that the immune reactive band is different from your predicted molecular weight, does it mean that I did not get the right over-expression lysate for my gene?
The predicted the molecular weight is calculated from the protein primary amino acid composition. There could be some discrepancy from the actual molecular weight, if there were post-translational modifications or known proteolytic activity. In this case, OriGene strongly suggests researchers check related literatures on this particular protein. Also refer to question 13 for reasons on why you can get multiple bands.
11. If the SDS samples have been frozen, do I have to re-boil them before loading?
Yes, it is recommended. It will help to denature the protein better.
12. I am working on a multi-span transmembrane protein. Should I boil my sample before SDS-PAGE fractionation?
No. According to our experience, the boiling of transmembrane protein in SDS sample buffer tends to induce protein aggregation. We suggest that the customer incubate the over-expression lysates with 1xSDS sample buffer at room temperature for 30 minutes before loading.
13. Why sometimes I see more than one band with some lysates in Western blot experiments?
There are multiple causes for the appearance of multiple bands in Western Blot. Here are possible causes and solutions:
|Excessive amount of lysate loaded||Dilute the lysates and determine the optimal loading amount.|
|Multiple low molecular weight bands might be the result of protein degradation||Use fresh lysates. Avoid multiple freeze-thaw cycles of cell lysates. Aliquot in small volumes.|
|Proteolytic breakdown of the target protein||Add a broad range protease inhibitors to inhibit all proteases.|
|Insufficient blocking||Try a variety of blocking agents.|
|Aggregation of target protein||Some proteins tend to aggregate and sometimes cannot be resolved by SDS and boiling.|
|Post-translational Modifications||Some post-translational modifications cannot be resolved by SDS and boiling.|
|Non-specific binding of primary/secondary antibodies||
Try a range of blocking agents including 2% non-fat dry milk.
Decrease primary/ secondary antibody concentration.
Add detergents Tween 20 to the antibody solutions.
Increase salt concentration in blotting and washing buffers.
14. How should I cite your product?
Here is our guideline:
Full Product Name, OriGene Technologies Inc., Rockville, MD, USA, Catalog #, Lot #.
In addition, we would love to hear from you when the paper is published. Email us a copy of the accepted manuscript and receive a special gift.