Urgent if anyone could help me check my answers and elaborat

Urgent!! if anyone could help me check my answers and elaborate that would be sooo helpful!

I\'m more confused on this one..If we have low specific activity that means we don\'t have our protein of interest purified. What should we conclude from this experiment? If you could be detailed, i\'d really appreciate it

On this one I\'m assuming that we cannot conclude anything about Km because we cannot even conclude anything about Vmax. What else is this question looking for and am I right that we can\'t conclude anything about Km?

You take total cell lysate, and use immunoprecipitation to purify an enzyme of interest. The purified enzyme has HIGH specific activity, and shows a single band (56kD) on an SDS PAGE. Now, you heat the total cell lysate, and use immunoprecipitation to purify the enzyme of interest. The purified enzyme has LOW specific activity, and shows two bands (56kD and 122kD on an SDS PAGE. You then heat the total cell lysate, allow it to cool down, and use immunoprecipitation to purify the enzyme of interest. The purified enzyme has HIGH specific activity, and shows one band (56kD) on an SDS PAGE. Finally, you make total cell lysate from a mutant cell. When you use immunoprecipitation to purify the enzyme of interest, the purified enzyme has LOW specific activity, and shows a single band (56kD) on an SDS PAGE. When you heat the cell lysate from the mutant cells and immunoprecipitate the enzyme, the purified enzyme has Low specific activity, and shows a single band (56kD) on an SDS PAGE. What is the BEST conclusion from this experiment? Explain your answer.

Solution

in your first condition when you heated the cell lysate and did immunoprecipitation with antibody. there might be the chances that the 122 kD protein get denatured and exposed same type of epitope which antibody recognizes in your protein of interest. that\'s why you are getting two bands in SDS page. also your protein shows low specific activity because it gets denatured with heat treatment (but not 100% gets denatured).

now in second case you heat the sample and then cool down before immunoprecipitation (IP). because of cooling it down your protein gets renature and the other (122kD) too. so there will be no epitope present on the 122 kD protein which your antibody recognize.that\'s why you got single band on SDS PAGE and high specific activity also.

in third case you are not very clear. mutant cell of what....... but if 56 kD protein mutant cell, then might be the protein forms aggregate and you ended up with low specific activity. though you will get a single band of 56 kD in SDS PAGE under REDUCING condition...... so my suggestion try to run SDS PAGE non reducing (without beta mercpto ethanol containing) and with reducing dye. in both the condition heating or non heating you will get low specific activity.

in your graph question you are talking about isozymes. the graph represent how enzyme works (by lowering the activation energy). you can not conclude anything about Km and Vmax....for this you need reciprocal plot or double reciprocal plot of enzyme kinetics.(Lineweaver-Burk plot or sigmoidal curve).

hope this helps.