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Cell Lab

Satisfactory Essays
Cell Lab

Materials and Methods and Results for lab 4.

Materials and Methods

Homogenization

Spinach leaves were used. The large veins were removed and the sample was weighed. Four grams were used to start. The leaves were chopped finely and added to an ice-cold mortar with 15 ml of homogenization buffer( .33M sorbitol, .05M HEPES, 4mM MgCl2, 2mM EDTA, pH 6.5).

The leaves were grinded to a paste and filtered through two layers of cheesecloth. The filtrate was collected, and each lab group was given a 1.5 ml aliquot of the filtrate. We took a 100uL of this solution and put into a microfuge tube labeled “C”.

Centrifugation

The microfuge tube was set aside on ice while the remaining filtrate was centrifuged at 200g’s.

The supernatant was removed and 100ul were set aside in a tube labeled “LSS” (for low speed supernatant) on ice. The remaining supernatant was centrifuged at 1000g’s for 7 minutes. 100 uL were taken from supernatant, and placed in a microfuge tube on ice labeled “HSS.” The pellet was resuspended in 250 ul of homogenization buffer. 100 uL of this solution was set aside in a tube labeled “HSS.”

SDS-PAGE preparation.

Eight microfuge tubes were taken and divided into two sets of four, labeled A and B. Each set had was labeled like the four we prepared earlier, “C”, “LSS”, “HSS”, “HSP.” 25 ul of the respective solution was pipetted into the two sets of tubes. Set A was given 25ul of 2X Laemli buffer A. Set B was given 25 ul of Laemli buffer B. Laemli buffer ( .125M Tris HCl, pH 6.8, 20% glycerol, 4% SDS, and 10% BME). One of the two buffers was made without BME, which breaks disulfide bonds. The buffer without BME will be determined by SDS-page analysis.

The samples were boiled for 5 min, and stored at 20 degrees C for one week.

Protein quantification

Five tubes labeled “Blank”, “C”, “LSS”, “HSS”, “HSP” were obtained. The blank contained 100 ul of distilled water. The others had 90 ul of distilled water and 10 ul of the cellular fractions obtained by centrifugation. #ml of BCA were added. The samples were placed in a water bath and incubated at 37 degrees Celsius for 30 min.

The incubated samples were placed in the model 340 spectrophotmeter and read at 562 nm. The readings were taken, and the protein amounts were calculated using a standard curve( y = .
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