What is the volume of B-PER Extraction Reagent to use per gram weight of wet cells?
For 1 gram of cells add 4 ml of B-PER Reagent.
Are B-PER Reagent components compatible with commercially available 6xHis- and GST-tagged fusion protein purification columns?
B-PER Reagent is designed to be used with, among other applications, 6xHis and GST fusion protein purification. When using B-PER Reagent with other IMAC or glutathione columns, use a dilution of at least one-to-one B-PER Reagent in the binding buffer recommended for that column. This will decrease the possibility of incompatibility of buffer components.
Do cells need to be sonicated and/or frozen before treatment with B-PER Reagent?
B-PER Reagent is designed to replace sonication, which cannot fully recover soluble proteins or regain inclusion bodies. Although freezing the bacterial pellets is not usually necessary, it appears to increase protein yield in some bacteria that are traditionally difficult to lyse.
How can I measure the amount of protein I have extracted?
The Pierce BCA Protein Assay Kit (Product # 23225) works very well to detect proteins extracted with B-PER Reagent. The Coomassie Plus Protein Assay (Product #23236) can be used with B-PER Reagent protein extractions, however when using B-PER with PBS or B-PER II reagents, dilute the protein preparation two-to-four fold before assaying with Coomassie Plus, or prepare sample with Compat-Able Protein Assay Preparation Reagent Set (Product #23215).
How much B-PER Reagent should I use for large-scale preps?
Using 1 liter of E. coli culture with an OD600 ~2 will give ~8 g wet cells. For 8 grams of cells use 32 ml of B-PER Reagent.
How will I know if my recombinant protein is soluble or insoluble?
SDS-PAGE examination of the protein pellet, as well as the lysate, will reveal whether the protein of interest is present in inclusion bodies or if it is soluble.
My protein is insoluble. Can I purify it using B-PER Reagent?
Inclusion bodies are easily purified using B-PER Reagent. To recover your protein from inclusion bodies, however, we recommend the use of Pierce Inclusion Body Solubilization Reagent (Product # 78115). Inclusion bodies can also be solubilized with 8 M Guanidine HCl or 8 M Urea.
The bacteria do not lyse with B-PER reagent. What is the problem?
Bacteria grown in rich medium (high glucose) are difficult to lyse. Try reducing the glucose concentration. Bacteria and organisms lysed with B-PER Reagent include E. coli, Acetinobacter, Archaebacteria, S. aureus, H. pylori, Baculovirus, nematodes and insect cells. B-PER Reagent can lyse some gram positive bacteria, but tends to be more efficient with gram negative bacteria. If lysis is inefficient for a particular bacterial strain, freeze cells before extraction. Add lysozyme for the most efficient extraction, however, for some over-expressed proteins the addition of lysozyme is not required.
What is the composition of B-PER Reagent?
B-PER Reagent utilizes a proprietary, mild nonionic detergent in a 20 mM Tris HCl, pH 7.5 buffer. No enzymatic components are present. Depending on your particular protein, you may need to add components such as salt, lysozyme, protease inhibitors, reducing agents and chelating agents. Other ready-to-use formats include B-PER II (2X B-PER),
B-PER in Phosphate Buffer, B-PER with Enzymes and B-PER Direct with Enzymes.
What is the maximum amount of NaCl that I can add to B-PER Reagent for purification of my salt-dependent protein?
You can add up to a final concentration of 0.5 M NaCl.
What is the difference between B-PER and B-PER II Reagent?
B-PER II Reagent contains more detergent making it ideal for extracting proteins from small bacterial cultures with less than 20 ml in volume.
What are the advantages to using the B-PER Direct Extraction Kit?
The Tris-buffered detergent concentrate and enzymes are formulated to lyse bacteria directly in culture media. The detergents and enzymes work in conjunction to gently solubilize proteins and eliminate any particulates in the extract making this process amenable to rapid, high-throughput protein screening and purification. The B-PER Direct Protocol requires no centrifugation steps and is ideal for screening 96-well microplate samples. Lysates can be applied directly to affinity purification resins.
Where is the DNA found when extracting proteins with B-PER Reagent?
It is found in the pellet after the first round of extraction of soluble proteins, so it should not be present in the final protein preparation.
Why is my protein extract so viscous?
Will my protein be in its native conformation after B-PER Reagent extraction?
This is often an indication of the presence of large amounts of DNA in the extract. This viscosity will be greatly decreased upon addition of DNase I (Product # 90083).
While this is protein-dependent, many proteins have been successfully tested in downstream applications (reporter assays, immunoprecipitation and beta-Gal assays), including GST and 6xHis proteins. Otherwise, samples can be diluted or dialyzed to remove any interfering substances.