Describe a chromatographic method in terms of beads, flow rate , sample buffer, equilibrium buffer, wash buffer, elution buffer, pressure head, detector head for separation of tripsinogen from alpha chymotripsinogen? How you will optimize run solution?
Answers
Answer:
Explanation:
Basic Guide to Protein Chromatography/Purification
BASIC CHROMATOGRAPHY: The
chromatography tutorial has already introduced you
to the concepts of several chromatographic resins,
the application of selecting a separation technique,
the analysis of chromatography and the selection of
the samples to pool and move onto the next step of
purification. The information presented here is less
about theory (there are several links for this on your
laboratory webpage) and more about the practical
aspects of purification. This is vital information you will
not find in most textbooks.
As mentioned in the
introduction, there are six
basic steps in purification:
Step 1 - Design the
Chromatography: This is the
step that will make or break
most of your efforts. Too little
attention here will result in frustration when preparing
solutions and running the column. Take the time to be
thorough in this step. There are several components
that need to be considered when designing your
chromatographic purification: the type of separation
technique, sample preparation, size of column, flow
rate, buffers needed to bind proteins, wash unwanted
proteins and elute the desired protein.
Before choosing a separation technique, you should
review the purification strategy found in the tutorial
and focus on the choices of purification methods. In
short, you should try to avoid using a chromatography
method that uses the same chemistry to separate
compounds. In other words, you will typically get poor
yield and purification from two ion exchange columns
or two size exclusion columns in a row, one right after
the other. You should also consider how the sample is
eluted from the column. Will the final buffer be
amenable to directly load onto the next column or will
you have to prepare the pooled fractions by dialysis or
concentrate them by ammonium sulfate fractionation
before continuing on?
Once you’ve picked your purification resin, you need
to determine the size of column, volume of resin, how
to load the sample, flow rate (how fast to run the
column), how much buffer to run through the column,
how to elute the sample and the collection method.
You will be given three or four different
chromatography resins to chose from. In the protocol
section on the class webpage, there are basic
protocols to help with each step. You will be
given the specifics on how to address the
important questions listed above in these
protocols. You should be aware of the
advanced theory and practice of each
chromatography. There are many excellent
websites which have this information, your class
website has a few. Chapter 11 in Principles and
Techniques of Biochemistry and Molecular
Biology (6th Ed.) has much of this key information
for you to look over. However, there are a few
basics that you should understand
before looking through the
protocols.
1) Column size – A short thick
column will have less backpressure
than a taller thin column (that is
how much resistance the fluid has
as it is pumped through the
column). Too much backpressure
and your tubing, connections and pump will fail.
A wide short column will have a faster flow rate.
If you are going to simply bind, wash, and elute
with what is called a different wash (step or
isocratic gradient) and not a gradual change
from one buffer to another (gradient elution)
then you might consider using a wider short
column. But you should also be aware that the
eluted sample will be more dilute and less
resolution (separation from contaminants). If you
are going to use a gradient elution or work with
size exclusion resins, then you must have a taller
thin column.
The specific amount of resin depends on the
type of chromatography and the particular
binding capacity of each resin. For resins that
bind their analyate, the top 20% of the column
should bind most of the protein, for SEC columns,
the sample volume loaded should be no more
than 3% of the bed volume (bed volume = the
volume of resin in the column).
2) Flow Rate: For simple open columns, gravity
will work just fine. If you are pumping buffers
through the column, then the flow rate can vary.
A fast flow rate may cause excess packing of the
resin and the backpressure will build, causing the
tubing and pump to leak and fail. At a high rate
of flow, most peristaltic pumps (the kind with a
tube stretched around a roller) will pulse back
Low
theoretical
plates, low
pressure, fast
but poor
resolution
High
pressures,
slower
flow rate,
high
degree of
resolution
between
proteins
Fig. Short wide vs. tall thin column
2
and forth, causing a dilution of the eluted proteins and
mixing of unresolved proteins. Too slow and the
proteins may lose activity waiting to elute. A simple
rule of thumb is set the flow rate no faster than 2 or 3
times that of gravity for soft resins (agarose and
sepharose) for 2-5 times that for more cross-linked
resins.