101407 To 102007
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Weekly Report by Huaming Fang
10/15/07----10/19/07
Label His-gp16 with Cy3-maleimide Aim: To label the cysteines residue of His-gp16 by Cy3-maleimide. Method: Conjugate the Cy3-maleimide onto Cysteine residues of gp16 by passive reaction, and then remove free dye by dialysis. And the antibody of His-gp16 was thought to be a positive control and used as the positive control. This time we used TCEP to reduce the –SH group. His-gp16(0.497mg/ml) Antibody of Hisgp16(1mg/ml) TCEP(3.22mM) 1X HMS contain 20% glycerin
1 5μl ---
2 --5μl
1μl 94μl
1μl 94μl
Incubate at cold room(4ºC) for 30min
Add 5μl Cy3-maleimide(in DMSO) and incubate at cold room overnight
Dialyze the sample in HMS (20%glycerin) for more than 20 hours
Measure the sample by UV
Result and discussion: The data of UV absorbance gave us a surprise (Figure 1). It seems that His-gp16 was labeled by Cy3-maleimide, because the UV absorbance is high at the 550 nm. However, the antibody of His-gp16, which was thought as a positive control, has a much lower UV absorbance at 550nm. But it is possible that the free dye was not removed by dialysis. To exclude the possibility, negative control which no proteins was added was done as the method mentioned above. After dialysis, most of free dye was removed. However, though its UV absorbance at 550nm is lower than His-gp16 sample’s, it is higher than antibody of gp16’s. So to make sure that His-gp16 is really labeled, next week we are going to dialyze longer, such as 36 hours.
Weekly Report by Huaming Fang
10/15/07----10/19/07
0.15
0.1 Absorbance (AU)
Cy3 His-gp16
0.05
antibody
0
-0.05 420
440
460
480 500 520 540 Wavelength (nm)
560
580
600
620
Figure 1: Cy3 means Cy3-maleimide negative control which no protein was added, and antibody means antibody of His-gp16.
Cy3 maleimide negative control before dilysis Cy3 maleimide negative control after dilysis His-gp16 labeled with cy3 maleimide after dilysis antibody of his-gp16 label with Cy3 maleimide after dilysis 1.8 1.6 Absorbance (AU)
1.4 1.2 1
0.8 0.6 0.4 0.2 0
-0.2 420
440
460
480 500 (nm) 520 Wavelength
Figure 2, it shows at least most free dyes are removed by dialysis.
540
560
580
600
620
10/15/07----10/19/07
Label His-gp16 with Cy3-maleimide Aim: To label the cysteines residue of His-gp16 by Cy3-maleimide. Method: Conjugate the Cy3-maleimide onto Cysteine residues of gp16 by passive reaction, and then remove free dye by dialysis. And the antibody of His-gp16 was thought to be a positive control and used as the positive control. This time we used TCEP to reduce the –SH group. His-gp16(0.497mg/ml) Antibody of Hisgp16(1mg/ml) TCEP(3.22mM) 1X HMS contain 20% glycerin
1 5μl ---
2 --5μl
1μl 94μl
1μl 94μl
Incubate at cold room(4ºC) for 30min
Add 5μl Cy3-maleimide(in DMSO) and incubate at cold room overnight
Dialyze the sample in HMS (20%glycerin) for more than 20 hours
Measure the sample by UV
Result and discussion: The data of UV absorbance gave us a surprise (Figure 1). It seems that His-gp16 was labeled by Cy3-maleimide, because the UV absorbance is high at the 550 nm. However, the antibody of His-gp16, which was thought as a positive control, has a much lower UV absorbance at 550nm. But it is possible that the free dye was not removed by dialysis. To exclude the possibility, negative control which no proteins was added was done as the method mentioned above. After dialysis, most of free dye was removed. However, though its UV absorbance at 550nm is lower than His-gp16 sample’s, it is higher than antibody of gp16’s. So to make sure that His-gp16 is really labeled, next week we are going to dialyze longer, such as 36 hours.
Weekly Report by Huaming Fang
10/15/07----10/19/07
0.15
0.1 Absorbance (AU)
Cy3 His-gp16
0.05
antibody
0
-0.05 420
440
460
480 500 520 540 Wavelength (nm)
560
580
600
620
Figure 1: Cy3 means Cy3-maleimide negative control which no protein was added, and antibody means antibody of His-gp16.
Cy3 maleimide negative control before dilysis Cy3 maleimide negative control after dilysis His-gp16 labeled with cy3 maleimide after dilysis antibody of his-gp16 label with Cy3 maleimide after dilysis 1.8 1.6 Absorbance (AU)
1.4 1.2 1
0.8 0.6 0.4 0.2 0
-0.2 420
440
460
480 500 (nm) 520 Wavelength
Figure 2, it shows at least most free dyes are removed by dialysis.
540
560
580
600
620
