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B-PER™ Direct Bacterial Protein Extraction Kit - FAQs

查看更多产品信息 B-PER™ Direct Bacterial Protein Extraction Kit - FAQs (90080)

10 个常见问题解答

细菌无法用B-PER试剂裂解，这是什么原因所致？

在富营养培养基（高糖）中生长的细菌往往难以裂解。您可以尝试减少葡萄糖浓度。B-PER试剂能够裂解包括E. coli，Acetinobacter，Archaebacteria，S. aureus，H. pylori，昆虫杆状病毒，线虫和昆虫细胞在内的细菌和细胞。B-PER试剂可以裂解一些革兰氏阳性菌，但是裂解革兰氏阴性细菌的效率通常更高。如果对某一特定细菌菌株裂解效果不佳，则请在裂解前冷冻细菌。添加溶菌酶是最有效的提取手段，但是对于一些过表达蛋白则不需要加入溶菌酶。

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.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

I lysed my cells with B-PER reagent, but my protein is denatured. How can I get functional protein?

Many proteins spontaneously refold into their native, functional structures when the denaturing solubilization reagents are removed by dialysis. Other proteins, however, will fold into non-functional and even insoluble forms by this process. In such cases, specialized sets of buffer conditions must be tested to identify those that promote the highest possible yield of properly refolded protein. See our Pierce Protein Refolding Kit (Cat. No. 89867).

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

My cells were lysed with B-PER reagent, but now the sample is very "sticky" or viscous. What can I do?

Add DNase I to the B-PER solution. If DNase was added, the enzyme may have low activity or be inactive. You can add Mg2+ ions to 2 mM final concentration, add more DNase I to the reagent, or purchase new enzyme. Alternatively, use B- PER Complete reagent, an all-in-one formulation that combines a lysis reagent with lysozyme and a universal nuclease to enable mild extraction from both gram-negative and gram-positive bacteria.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

I added enzyme to the B-PER reagent, but my bacteria were not lysed. What can I do?

The temperature may be too low; warm the culture and other reagents to room temperature. The lysozyme may have low activity or be inactive. Add more lysozyme to the reagent or purchase new enzyme.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

My bacterial cells were not lysed upon addition of B-PER reagent. How can I increase the lysis efficiency?

We recommend using B-PER Complete Bacterial Protein Extraction Reagent, an all-in-one formulation that combines a lysis reagent with lysozyme and a universal nuclease to enable mild extraction from both gram-negative and gram- positive bacteria. If the addition of lysozyme and DNase I might interfere with your downstream application, we recommend using other B-PER reagents. See details of the various B-PER reagents we offer.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

I lysed my cells in B-PER reagent, but my protein of interest wasn't solubilized. What can I do?

Some overexpressed proteins can be insoluble, misfolded, or expressed in inclusion bodies. The best way to obtain soluble protein is to adjust the expression conditions; however, if this is not possible, the Inclusion Body Solubilization Reagent (Cat. No. 78115) can be used.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

Which formulation of B-PER bacterial cell lysis reagent is right for me?

See details of the various B-PER reagents we offer (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/cell-lysis-fractionation/cell-lysis-total-protein-extraction/b-per-bacterial-cell-lysis-reagents.html).

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.

What lysis buffers do you offer for bacterial cells?

We offer the B-PER Bacterial Protein Extraction Reagent for lysis of bacterial cells.

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.