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Powdered Milk Lab Report

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At first, we prepared a 5 % fresh milk, that is for 30 mL PBS we added 1.5 g of powdered milk. Then we removed membrane from the “sandwich” and placed it on the cutting board, and folded plastic wrap around membrane. The size of the target protein is 100 kDa, so we cut it on 150 kDa and below 75 kDa. After removing plastic wrap and labeling the membrane, we put it in the 5% milk and placed it on the bench-top for 30 minutes agitation at room temperature. Powdered milk also contains many proteins, which coat the surface of the membrane. The coating of the membrane surface block our antibody from binding non-specifically to other proteins. After 30 minutes of agitation, membrane was rinsed 3 times in 1X PBST with gentle shaking, using a generous amount of buffer. Additionally, excess milk was removed by washing it 3 times…show more content…
We washed off the primary antibody from a membrane at the same manner as we washed off the milk, and then incubated the blot for an hour at room temperature with the secondary antibody GAR680, from this point a membrane was shielded from the light until it has been imaged. The secondary antibody specifically recognizes the primary antibody. GAR680 is Goat Anti-Rabbit immunoglobin antibody. This is an antibody raised in goats that recognizes rabbit immunoglobin. Excess secondary antibody is removed by washing in PBST buffer as was done for the primary antibody, the final wash with 1X PBS to remove residual detergent Tween 20. The membrane was ready for scan and reading on Li-Cor machine. Specially designated tray for fluorescent antibodies was cleaned up with deionized water and kimwipes, then only the membrane was placed on it and entered the Li-Cor machine. The membrane position was from the first to the 10th well, from left to the right, the label was on the left side. The membrane reading was made on 700 nm
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