The loading buffer for SDS-PAGE gels can be purchased in two forms – with or without β‑mercaptoethanol. If you were loading the gel with subtilisin (amino acid sequence given below), which version of loading buffer could you use? Can you explain why?MRSKKLWISLLFALTLIFTMAFSNMSAQAAGKSSTEKKYIVGFKQTMSAMSSAKKKDVISEKGGKVQKQFKYVNAAAATLDEKAVKELKKDPSVAYVEEDHIAHEYAQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAGTIAALNNSIGVLGVAPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVDKAVSSGIVVAAAAGNEGSSGSTSTVGYPAKYPSTIAVGAVNSSNQRASFSSAGSELDVMAPGVSIQSTLPGGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ
Question
The loading buffer for SDS-PAGE gels can be purchased in two forms – with or without β‑mercaptoethanol. If you were loading the gel with subtilisin (amino acid sequence given below), which version of loading buffer could you use? Can you explain why?MRSKKLWISLLFALTLIFTMAFSNMSAQAAGKSSTEKKYIVGFKQTMSAMSSAKKKDVISEKGGKVQKQFKYVNAAAATLDEKAVKELKKDPSVAYVEEDHIAHEYAQSVPYGISQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLNVRGGASFVPSETNPYQDGSSHGTHVAGTIAALNNSIGVLGVAPSASLYAVKVLDSTGSGQYSWIINGIEWAISNNMDVINMSLGGPTGSTALKTVVDKAVSSGIVVAAAAGNEGSSGSTSTVGYPAKYPSTIAVGAVNSSNQRASFSSAGSELDVMAPGVSIQSTLPGGTYGAYNGTSMATPHVAGAAALILSKHPTWTNAQVRDRLESTATYLGNSFYYGKGLINVQAAAQ
Solution
The choice of loading buffer for SDS-PAGE gels depends on the nature of the protein being loaded. In this case, the protein is subtilisin, which is a serine protease.
The purpose of β-mercaptoethanol in the loading buffer is to reduce disulfide bonds in proteins. This is important for proteins that have disulfide bonds as it allows them to fully denature and run properly on the gel.
Looking at the amino acid sequence of subtilisin, it does not contain any cysteine residues. Cysteine residues are the amino acids that form disulfide bonds. Therefore, subtilisin does not have any disulfide bonds that need to be reduced.
So, in this case, you could use the loading buffer without β-mercaptoethanol for the SDS-PAGE gel. The absence of β-mercaptoethanol will not affect the denaturation of subtilisin or its migration on the gel.
Similar Questions
Pre-lab Question, Session 2: Which of the following is a consequence of adding SDS to sample buffer and the SDS gel, adding beta-mercaptoethanol to the sample buffer, and boiling the sample before running our SDS-PAGE gel in lab?Group of answer choicesProteins can be separated based on sizeAll of these answers are correctProteins can be separated based on their 3D structureProteins can be separated based on their net charge
A protein was run under native conditions in a gel filtration column and was calculated to be of 120 kDa in size. The molecular weight of the protein in SDS-PAGE was found to be 30 kDa. The oligomeric status of the protein is
When performing a Western blot, after running an SDS-PAGE gel, a transfer step is performed.The purpose of this transfer is to:Group of answer choicesAllow the proteins to be visualisedReduce the background ‘noise’ and reduce false positive resultsImmobilise proteins onto a nitrocellulose membraneSeparate the proteins based on their size and charge
Which of the following property of a protein is exploited in gel filtration chromatography
Decreasing the ratio of acrylamide:bisacrylamide when pouring an SDS-PAGE gel makes the gel "thicker" (less porous). Can you explain why?
Upgrade your grade with Knowee
Get personalized homework help. Review tough concepts in more detail, or go deeper into your topic by exploring other relevant questions.