SMART Digest™ 免疫亲和 (IA) 蛋白质 G 试剂盒

We have observed the following effects with detergents:
• CHAPS – ~ 30% reduction in digest efficiency
• OGS – No reduction in digestion efficiency
• TWEEN – No reduction in digestion efficiency
• RIPA – The addition of RIPA, for ribonuclease A digestion, results in a concentration-dependent effect, where initial enzyme inhibition is overcome by improved substrate solubilization at higher concentrations only. 20% reduction in digestion efficiency.

We recommend compensating for reduction in digestion efficiency by extending the digestion time accordingly.

The SMART Digest kits are not affected by concentrations of up to 0.5M of urea. If the concentration is higher than this, we recommend that the sample is diluted to <0.5 M of urea, prior to beginning digestion using the SMART Digest kit.

The composition of the SMART Digest IA buffer is as follows:
Chemical Name (CAS No., EINECS No.): Kit Component Qty, Weight %
• Water (7732-18-5, 231-791-2): 2, 50-95%
• Glycerol (56-81-5, 200-289-5): 2, < 20%
• Tris Base (77-86-1, 201-064-4): 2, < 10%
• Tris-HCI (1185-53-1, 214-684-5): 2, < 10%
• Calcium Chloride (10043-52-4, 233-140-8): 2, < 10%
• Sodium Azide (26628-22-8, 247-852-1): 2, < 0.1%

The SMART Digest IA Kits contain an excess of enzyme capable of digesting between 200 pg and 3.5 mg of protein. As most samples will fall within this range, it is not necessary to routinely quantitate protein concentration prior to digestion.

The reason urea is needed as a first step in an in-solution protocol is to disrupt the sample and partially unfold the proteins. The proteins need to be partially unfolded so that the trypsin enzyme can have better access to the internal amino acid chain, not just the surface of the protein of interest. The reason the SMART Digest IA kits do not need urea is that it uses heat to unfold the protein.