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A critical step in performing any biological assay is preparing the test sample. Antibody-based ELISA and dipstick assays capture sample proteins and protein complexes in their native, non-denatured state. Many assays, notably our OXPHOS assays, capture membrane-bound multi-subunit enzymes. Sample proteins for these assays must be extracted by non-denaturing methods, not just for recognition by the capture antibodies, but also to retain their enzymatic function for activity assays. Therefore, we have developed assays using a non-denaturing detergent, n-Dodecyl-beta-D-maltopyranoside (CAS# 69227-93-6), which is commonly referred to as lauryl maltoside. Lauryl maltoside is a non-ionic detergent used for the stabilization and activation of enzymes in membrane research, and thus is well suited for the analysis of mitochondrial membrane proteins and enzyme complexes 1-7.
The guide outlines the steps to perform lauryl maltoside extractions of functional enzymes and proteins from tissues and cultured cells for use with our ELISA and dipstick assays.
Many of our assays are reactive with rodent proteins and were developed with rodent tissues (mouse and rat). The data in this section of the guide was obtained with rodent tissues, but the procedure applies to human and other animal tissues as well. Fully functional enzymes can be extracted from tissues by following a few simple steps.
We use simple mechanical disruption with Dounce homogenizers to disrupt cells minimally. Tissue is homogenized sequentially with two different-sized pestles and processed until smooth enough to be pipetted. Sequential homogenization is started with a large-clearance pestle that provides a coarse disruption of the tissue and finished with a small-clearance pestle that provides a finer disruption of the tissue. A suitable Dounce homogenizer is available from us in our tissue sample preparation kit ab110169.
For long-term storage, it is best to make tissue homogenates that are as concentrated as possible. We have found that tissue can be homogenized with Dounce homogenizers with relative ease up to 25 mg/ml. After homogenization, sample detergent lysates can be made immediately or tissue homogenates can be aliquoted and frozen at -80°C.
|Tissue||Wet weight (mg/ml)||Buffer expected protein concentration (by BCA)|
Table 1: Typical wet weight starting guide
The amount of tissue homogenized per ml of buffer to make a homogenate that is approximately 25 mg/ml protein by BCA protein analysis.
Materials for tissue homogenization
Method for tissue homogenization
Protein extraction with lauryl maltoside is, in most cases, optimal with samples that contain a protein concentration of 5 mg/ml.
To extract protein from the prepared tissue homogenate, MitoSciences kits use one or two methods. We have noticed no significant difference in protein yield or assay performance between these two methods.
ATP synthase - For the Complex V microplate activity assay ab109704, it is recommended to thoroughly freeze/thaw the sample and wash the pelleted tissue homogenate to remove any soluble, non-membrane associated proteins. This is particularly important for liver preparations, which contain many non-membrane-associated proteins. Concentrating the membrane proteins before protein assay leads to an improved detergent solubilization step and maximizes enzyme stability and oligomycin sensitivity.
PDH - For the PDH microplate activity assay ab109902, it is recommended to add the supplied detergent to a protein concentration of 25 mg/ml (undiluted homogenate) in order to maintain enzyme stability. Extracts should be centrifuged at a low speed (1,000 x g).
Materials for protein extraction
Protein extraction method I with 1X extraction buffer:
Protein extraction method II with 10X lauryl maltoside:
The protein concentration of the extract should now be ≤ 5 mg/ml, depending on extraction efficiency, as measured by BCA assay. To measure protein concentration with a NanoDrop™ spectrophotometer, follow the maufacturer's instructions for measuring protein and in the pull-down menu for sample type, select "other protein (E 1%)", then enter an extinction coefficient (Ext. Coeff, E 1% l/gm-cm) from table 2 below.
|Cell line||Extinction coefficient|
Table 2: Extinction coefficients for cell extracts
It may be possible to freeze the extract at -80°C before running the assay without significant loss of activity.