2-D Quant Kit
2-D Quant Kit is designed for the accurate determination of protein concentration in samples prepared for electrophoresis techniques such as 2-D electrophoresis, SDS-PAGE, or IEF.
FAQ
The BSA standard curve is flatter than the example given in the instructions. How should I proceed?
Figure 1. Typical standard curve.
Determine the nature of the flatness. Does the standard curve start as expected, up around 0.75 or above (curve A above)? Or does it start out very low (curve B above)?
Curve A. Y-intercept is normal but slope is low.
1. Color reagent is not being introduced to the tube or well by instantaneous mixing. Color reagent should be introduced by rapidly shooting it into the tube.
2. Less BSA standard is being added than Customer A thinks.
a. Improper precipitation and resolubilization of the BSA standard. Skip the precipitation steps for the BSA standard curve! (There is never any need to precipitate the standard BSA before assay.)
b. Examine the BSA standard for any signs of precipitation. Make sure the BSA was stored properly. How old is it?
c. BSA is degraded. Again, make sure it was stored properly and not contaminated.
d. If Customer A has any lyophilized BSA present, have Customer A prepare a separate BSA standard and make a standard curve using Customer A’s own BSA solution. Is this curve any different?
e. Pipetting errors. Also make sure the pipetter is calibrated properly.
3. Something in the BSA solution is interfering.
a. Do not add anything to the stock tube of BSA standard.
B. Curve B. Both Y-intercept and slope are low. In other words, the curve starts near the end of the linear range, so it’s no surprise that the slope of the curve is flatter.
1. Less copper solution is present from the start.
a. Pipetting errors. Also make sure the pipetter is calibrated properly.
2. More BSA standard is being added than Customer B thinks. Perhaps even present in the color reagent from the start.
a. Pipetting errors. Also make sure the pipetter is calibrated properly.
b. Contamination of the assay tubes or color reagent.
3. Interfering substance present in the water that is added with the copper solution. Only use the purest water available, at least distilled or deionized.
4. Spectrophotometer errors
a. Make sure the spec is set to read at 480 nm. If not, then it is not reading at the maximum absorbance, and will therefore give anomalously low readings. (Have the customer spin the dial around 480 nm to see if the Absorbance can increase. If the max is not when the dial is at 480 nm then the spec is out of calibration.)
How do I prepare immunoprecipitated proteins for DIGE labeling?
Immuno precipitated proteins can be removed from the beads at low pH with 100 mM glycine-HCl, pH 2.7. After neutralization with Tris base the sample can be desalted using the 2-D Cleanup kit. The sample is now ready for CyDIGE labeling using the standard protocol.
Troubleshooting
Find solutions to product related issues. For unlisted issues please contact local Cytiva service representation.
Select symptom:
| Possible cause | Suggested remedy |
|---|---|
Pellet not completely mixed with the color reagent (Step 9)* and protein is thus slowly entering solution in the cuvette, binding more copper and lowering absorbance over time. *Steps refer to the procedures below, Standard procedure and Procedure for Ettan™ DIGE |
Ensure instantaneous mixing by introducing the working color reagent to the tube as rapidly as possible. Mix by inversion or vortexing as soon as possible after introducing the reagent. |
| Possible cause | Suggested remedy |
|---|---|
Insufficient centrifugation. |
Ensure that the protein is fully sedimented and forms a tight pellet by centrifuging for at least 5 min at a minimum of 10 000 x g. |
The working color reagent was not the sample (Step 9).* *Steps refer to the procedures below, Standard procedure and Procedure for Ettan™ DIGE |
Ensure instantaneous mixing by introducing the working color reagent to the mixed rapidly with tube as rapidly as possible. Mix by inversion or vortexing as soon as possible after introducing the reagent. |
| Possible cause | Suggested remedy |
|---|---|
The protein was not fully resuspended following centrifugation (Step 8).* *Steps refer to the procedures below, Standard procedure and Procedure for Ettan™ DIGE |
Some sample solution components may result in a protein pellet that is more difficult to resuspend. Vortex the tube (Step 8)* for at least 10 s |
| Possible cause | Suggested remedy |
|---|---|
Carry over of interfering reagents in the protein solution |
1. Ensure that the supernatant is completely removed following centrifugation (Step 7).* There should be no visible liquid remaining in the tubes. *Steps refer to the procedures below, Standard procedure and Procedure for Ettan™ DIGE |
| Possible cause | Suggested remedy |
|---|---|
Use of incorrect reference. |
Always use water as the reference. |
Change me
Standard procedure
1. Prepare a standard curve according to Table 1 using the 2 mg/ml Bovine serum albumin (BSA) standard solution provided with the kit. Set up six tubes and add standard solution according to Table 1. Tube 1 is the assay blank, which contains no protein.
Table 1. Preparation of standard curve
Note: The accuracy of the assay is unaffected by the volume of the sample as long as the sample volume is 50 μl or less. It is therefore unnecessary to dilute standard or sample solutions to a constant volume.
2. Prepare tubes containing 1–50 μl of the sample to be assayed.
Duplicates are recommended. The useful range of the assay is 0.5–50 μg and it is also recommended that more than one sample volume or dilution be assayed for each sample to ensure that the assay falls within this range.
3. Add 500 μl precipitant to each tube (including the standard curve tubes). Vortex briefly and incubate the tubes 2–3 min at room temperature.
4. Add 500 μl co-precipitant to each tube and mix briefly by vortexing or inversion.
5. Centrifuge the tubes at a minimum of 10 000 × g for 5 min.
This sediments the protein.
6. Remove the tubes from the centrifuge as soon as centrifugation is complete. A small pellet should be visible. Decant the supernatants. Proceed rapidly to the next step to avoid resuspension or dispersion of the pellets.
7. Carefully reposition the tubes in the microcentrifuge as before, with the cap-hinge and pellet facing outward. Centrifuge the tubes again to bring any remaining liquid to the bottom of the tube. A brief pulse is sufficient. Use a micropipette to remove the remaining supernatant. There should be no visible liquid remaining in the tubes.
8. Add 100 μl of copper solution and 400 μl of distilled or de-ionized water to each tube. Vortex briefly to dissolve the precipitated protein.
9. Add 1 ml of working color reagent to each tube (See “Preliminary preparations” for preparing the working color reagent). Ensure instantaneous mixing by introducing the reagent as rapidly as possible. Mix by inversion.
10. Incubate at room temperature for 15–20 min.
11. Read the absorbance of each sample and standard at 480 nm using water as the reference. The absorbance should be read within 40 min of the addition of working color reagent (step 9).
Note: Unlike most protein assays, the absorbance of the assay solution decreases with increasing protein concentration. Do not subtract the blank reading from the sample reading or use the assay blank as the reference.
12. Generate a standard curve by plotting the absorbance of the standards against the quantity of protein. Use this standard curve to determine the protein concentration of the samples.
Procedure for Ettan™ DIGE
We recommend the following procedure for 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE).
1. Prepare a standard curve according to Table 2 using the 2 mg/ml Bovine serum albumin (BSA) standard solution provided with the kit. Set up six tubes and add standard solution according to Table 1. Tube 1 is the assay blank, which contains no protein.
Table 2. Preparation of standard curve.
Note: The accuracy of the assay is unaffected by the volume of the sample as long as the sample volume is 50 μl or less. It is therefore unnecessary to dilute standard or sample solutions to a constant volume.
2. Prepare tubes containing 1–50 μl of the sample to be assayed.
Duplicates are recommended. The useful range of the assay is 0.5–50 μg and it is also recommended that more than one sample dilution be assayed for each sample to ensure that the assay falls within this range. When preparing dilutions it is important to dilute the sample in the same sample buffer used as for the original test sample to keep the amount of sample buffer added to the standard curve consistent.
3. Add 500 μl precipitant to each tube (including the standard curve tubes). Vortex briefly and incubate the tubes 2–3 min at room temperature.
4. Add 500 μl co-precipitant to each tube and mix briefly by vortexing or inversion.
5. Centrifuge the tubes at a minimum of 10 000 × g for 5 min. This sediments the protein.
6. Remove the tubes from the centrifuge as soon as centrifugation is complete. A small pellet should be visible. Decant the supernatants.
7. Repeat steps 3 to 6. Proceed rapidly to the next step to avoid resuspension or dispersion of the pellets.
8. Carefully reposition the tubes in the microcentrifuge as before, with the cap-hinge and pellet facing outward. Centrifuge the tubes again to bring any remaining liquid to the bottom of the tube. A brief pulse is sufficient. Use a micropipette to remove the remaining supernatant. There should be no visible liquid remaining in the tubes.
9. Add 100 μl of copper solution and 400 μl of distilled or de-ionized water to each tube. Vortex briefly to dissolve the precipitated protein.
10. Add 1 ml of working color reagent to each tube (See “Preliminary preparations” for preparing the working color reagent). Ensure instantaneous mixing by introducing the reagent as rapidly as possible. Mix by inversion.
11. Incubate at room temperature for 15–20 min.
12. Read the absorbance of each sample and standard at 480 nm using water as the reference. The absorbance should be read within 40 min of the addition of working color reagent (step 9).
Note: Unlike most protein assays, the absorbance of the assay solution decreases with increasing protein concentration. Do not subtract the blank reading from the sample reading or use the assay blank as the reference.
13. Generate a standard curve by plotting the absorbance of the standards against the quantity of protein. Use this standard curve to determine the protein concentration of the samples.