this protein marker standard should be used. Figure 1: SDS PAGE Gel of Murine Fi
ID: 162789 • Letter: T
Question
this protein marker standard should be used.
Figure 1: SDS PAGE Gel of Murine Fibroblast Cell Lysate samples. Lane 1, 5 µL protein marker added. Lane 2, sample 3 containing 5 µL of a 16 µL of cell lysate solution. Lane 3, containing of 5 µL of a 9.6 µL cell lysate and 6.4 µL distilled water dilution solution. Lane 5, containing 5 µL of an 8 µL Bovine Serum and 32 µL distilled water solution. Lane 6, containing 5 µL of a 4 µL Bovine Serum and 16 µL of distilled water solution. Lane 7, containing 5 µL of a 2 µL Bovine Serum and 8 µL of distilled water solution. Lane 8, containing 5 µL of an 8 µL Horse Serum and 32 µL distilled water solution. Lane 9, containing 5 µL of a 4 µL Horse Serum and 16 µL of distilled water solution. Lane 10, containing 5 µL of a 2 µL Horse Serum and 8 µL of distilled water solution.
used: polyacrylamide gel is 0.1% Coomassie Blue dye in 50% methanol, 10% glacial acetic acid. We destained starting with 50% methanol, 10% acetic acid for 1-2 hours, then used 7% methanol, 10% acetic methanol to finish.
From left to right: Lane 10, Lane 9, Lane 8, Lane 7, Lane 6, Lane 5, Lane 4, Lane 3, Lane 2. Lane 1
Lab: SDS PAGE Gel of Murine Fibroblast Cell Lysate samples.
1. Discuss how one could improve there SDS PAGE Gel technique?
2. explain what these results show?
3. what would cause SDS PAGE Gel bands to be unclear? too thick? too fuzzy?
4. which band gave best result and why? does it relate to the serum used and its components?
Explanation / Answer
1.
The running behaviour of the gel indicating many faults in the prepration
A high quality acrylamide and bis need to be used and there ratio also need to be maintained accurately.
PH of the Tris and running buffer also play a important role in running behavior so that should be accurate.
For proper stacking we can increase salt concentration in stacking gel.
The Voltage should be optimized
2.
Lane 2,3
There is need to load more lysate and the bands are not sharp so sample preparation need to be improved.
Lane 5, 6 and 7 the dilution indicating the amount loaded in these three lane is same but it is not reflecting in the gel means unequal and overloading. the sharpness of thre band are not good all the bands are distorted.
Lane 8,9 and 10 the dilution indicating the amount loaded in these three lane is same but it is not reflecting in the gel means unequal and overloading. the sharpness of thre band are not good all the bands are distorted.
3.
The overloading makes the bands too thick because of that the bands are distorted and ugly.
4.
10th band giving the shapest band because it seems there the loading is less and the pattern is good.
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