Question

Outline the major steps for how TLR 4 might activate SAPK pathway.

Outline the steps necessary for preparing a cell lysate

The instructions in a protocol indicate that a 1/10,000 dilution of a stock solution is to be made in water. This means 1 part of stock solution and 9,999 parts of water. If you equate 1 part as 1 ml, this would mean that we would end up with 10,000 ml (= 10 liters) of the diluted solution which is a LOT of solution. Most probably, this total volume is impractical for your lab since you may not have enough material to make 10 liters or you may not have enough shelf space to store this amount. Furthermore, you may actually need only a few mls of the diluted solution for your experiment. Considering this, suggest an alternative route for diluting your stock that is quick and less wasteful.

A test on a urine sample was ordered. The concentration of the substance in the urine was too high to be determined with our instruments. We made a 1/10 dilution and ran the test on the diluted solution. The answer obtained was 50 mg/ml. What should be reported as the concentration of the substance in the undiluted urine?

Answer 1

A. Stress activated protein kinases ( SAPK) amino - terminal kinases (JNK) are members of the MAPK family and are activated by a variety of environmental stresses, inflammatory cytokines, growth factors and GPCR agonists.

B)

1. Harvest cells from bacterial culture by centrifugation ( 5000 rpm for 10 minutes ). Aspirate the supernatent and freeze the resulting pellet at -70^oC .
2. Resuspend the pellet/ bacterial cells in 2 ml MQ grade water and transfer the mixture to a clean tube.
3. Add lysozyme and incubate on ice for 30 minutes at 30 ^o C for 15 minutes or until the mixture becomes viscous.
4. Sonicate the sample on ice using three 10 second bursts at high intensity and let the mixture coll down for 30 seconds on ice between each burst. At the end of this step the sample should lose its viscosity as the DNA is sheared.
5. Using the prepared lysis buffer dilute the lysate to 50ml. Centrifuge at 35,000 rpm for 30 minutes at 4^oC to pellet the remaining cellular debris in the lysate. Transfer the supernatent to new tubes.
6. If there are any insoluble proteins in the pellet prepare a denatured lysate followed by denatured purification protocol to recover them.
7. Remove 5 micro litre of the lysate for SDS PAGE analysis and store remaining lystae on ice.

C. Divide the needed by dilution factor to determine unit volume of the concentrated urine sample.

D. 1 ml stock/ 10 ml water x 1/10x1/10x1/10 = 1/10,000 dilution. This means taking only 1 ml of the original stock and diluting it 1/10 four times. This would produce 10 ml of 1/10,000 dilution of stock water.