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Comparison of Buffer Exchange Techniques for GST Protein, Study Guides, Projects, Research of Biochemistry

An overview of three common buffer exchange techniques for glutathione s-transferase (gst) protein: dialysis, desalting column, and spin column methods. Each technique has its advantages and disadvantages, such as gentleness towards the protein, speed, cost, and ease of sample recovery. The document also includes a protocol for using spin columns for buffer exchange.

Typology: Study Guides, Projects, Research

Pre 2010

Uploaded on 08/18/2009

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koofers-user-rzb 🇺🇸

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Changing buffers for your GST.
1. Dialysis
Tubing with pores that have size smaller than the protein but larger than the
buffer contents.
Advantages
Gentle on the protein
Inexpensive
Can be done in cold room
Not labor intensive
Better for larger volumes
Disadvantages
Takes time. Couple days is best for complete buffer change
Risk of losing sample while handling tubing
Sample difficult to recover if lost through tubing or clips
2. Desalting Column
Use size exclusion resin. Sephadex G25, or pre-packed PD-10 columns
Advantages: Quick
Disadvantages: incomplete desalting
Not best for changing to a new buffer
More expensive
Dilutes protein
3. Spin Column
Attach a micro column to an eppi tube
Place sample in column and spin
Buffer passes through membrane, protein is retained in column cup
Advantages
More complete than desalting if repeated serially
Relatively fast
Can also concentrate the protein at the same time
Sample easily recovered if leakage occurs
Disadvantages
Limited volumes can be done at a time
Expense
Serial spins may require some time depending on cutoff size
Risk of drying sample, risk of precipitation of protein
Buffer inside
beads
GST passes around
beads and elutes
first without salts
pf2

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Changing buffers for your GST.

1. Dialysis Tubing with pores that have size smaller than the protein but larger than the buffer contents. Advantages Gentle on the protein Inexpensive Can be done in cold room Not labor intensive Better for larger volumes Disadvantages Takes time. Couple days is best for complete buffer change Risk of losing sample while handling tubing Sample difficult to recover if lost through tubing or clips 2. Desalting Column Use size exclusion resin. Sephadex G25, or pre-packed PD-10 columns Advantages: Quick Disadvantages: incomplete desalting Not best for changing to a new buffer More expensive Dilutes protein 3. Spin Column Attach a micro column to an eppi tube Place sample in column and spin Buffer passes through membrane, protein is retained in column cup Advantages More complete than desalting if repeated serially Relatively fast Can also concentrate the protein at the same time Sample easily recovered if leakage occurs Disadvantages Limited volumes can be done at a time Expense Serial spins may require some time depending on cutoff size Risk of drying sample, risk of precipitation of protein Buffer inside beads GST passes around beads and elutes first without salts

We have the spin columns. These other options are possible if you find that your conditions need them for your project. We can order these things if you give us advance notice. Protocol for the spin columns

  1. Add 300 μl of buffer to microcentrifuge tube. Place empty column (cup) into the tube.
  2. Add 400 μl initial sample to the column.
  3. Spin 5000 g for 20 minutes (approximately). Once spun long enough, your sample should come to a final volume of 40 μl. When you resuspend with new buffer, calculate dilutions to figure out if you have the desired concentrations of buffer. Ex. If you dilute your 40 μl retentate with 360 μl of new buffer. The old buffer is still present in 1/10 original concentration. If this is acceptable, you are done. If not, you made need to spin and dilute again.