1 |
Improper protein transfer to membrane |
- Stain gel after transfer is complete to determine transfer is efficient
- Use Ponceau S to stain membrane to determine transfer is efficient
- Ensure sufficient contact between gel and membrane during transfer
- Make sure transfer sandwich is assembled correctly
- Wet membrane according the instruction
- Avoid overheating during electro-transfer
- Use positive control or molecular weight markers
- Optimize transfer time and current
- Use Boster’s Membrane-Transferring Buffer (AR1149)
- Avoid sample (antigenic determinant) destroy when handling
|
2 |
Insufficient protein and membrane binding |
- Adding 20% methanol to transfer buffer
- Use small-bore membrane
|
3 |
Insufficient antibody |
- Increase antibody concentration
|
4 |
Insufficient antigen |
|
5 |
Antigen masking by blocking buffer |
- Compare different blocking buffers
- Optimize protein concentration of blocking agent
- Reduce blocking time
- Nonfat dry milk can sometimes mask antigens. Try using a different blocking reagent.
|
6 |
Presence of sodium azide in buffers |
- Eliminate sodium azide from buffers
|
7 |
Too short exposure time |
- Lengthen film exposure time
|
8 |
Too short substrate incubation time |
- Lengthen substrate incubation time to five minutes
|
9 |
Digestion of protein on membrane |
- Optimize amount of blocking agent
|
10 |
Degradation of protein during storage |
- Re-prepare protein sample
|
11 |
Incompatible primary and secondary antibodies |
- Make sure primary antibody, secondary antibody, substrate, enzyme system and samples are compatible
- Use loading control to test effectiveness of second detecting system
|
12 |
Low concentration of primary antibody and/or secondary antibody |
- Increase antibody concentration
- Increase incubation time
- Use a dot blot assay to optimize protein concentration
|
13 |
Cross-reactivity between blocking agent and antibodies (primary or secondary) |
- Use mild detergent such as Tween20
- Change blocking agent (commonly used are milk, BSA, serum or gelatin)
|
14 |
Inability of primary antibody to recognize the protein in tested sample |
- Check instruction
- Use positive control
|
15 |
Low or none content of target protein (ineffective antigen) |
- Use positive control
- Increase loading amount to 20-30 µg protein per well
- Use protease inhibitor or fractional extract target protein
|
16 |
Insufficient transfer and excessive wash |
- Check the transfer with Ponceau S
- Soak PVDF-membrane in methanol
- Avoid excessive wash
|
17 |
Over-blocking |
- Use 0.05% skim milk or no milk diluents buffer
- Change blocking agent
- Reduce blocking time
|
18 |
Loss of primary antibody effectiveness |
- Prepare fresh antibody and store properly when not in use
- Avoid repeated freezing and thawing
|
19 |
Inhibition of secondary antibody by sodium azide |
- Avoid using sodium azide together with HRP- conjugated antibodies
|
20 |
Loss of effectiveness in enzyme conjugate and substrate |
- Mix enzyme conjugate and substrate (no color development when enzyme is inactive)
- Use activated enzyme conjugate and fresh substrate
|
21 |
Improper wet transfer for membrane |
- Soak PVDF membrane in 100% methanol
|
22 |
Insufficient molecular weight of target protein (< 10 kDa) |
- Use small-bore membrane
- Reduce transfer time
|
23 |
Equality or nearness in values between target protein’s isoelectric point and transfer buffer’s pH value |
- Try other buffers such as CAPS buffer (pH 10.5)
- Try low pH value buffers such as acetic acid buffer
|
24 |
Too high methanol concentration |
- Decrease methanol concentration or use isopropyl alcohol
|
25 |
Insufficient sample concentration |
- Increase the amount of starting material
- Concentrate your sample using immunoprecipitation or similar procedure
|
26 |
Transfer too vigorous |
- Reduce transfer time or voltage to prevent small proteins transferring completely through membrane
- Use a secondary membrane to capture proteins transferred through the primary membrane
- Use a membrane with smaller pore size
|
26 |
Inadequate transfer |
- Increase transfer time or voltage
|
27 |
Sandwich assembly oriented incorrectly |
- Make sure the sandwich assembly is oriented correctly relative to the electric field
- Check the polarity of the electric field
|
28 |
Incorrect transfer buffer pH |
- Adjust transfer buffer PH to be 2 points lower than the pI of protein sample to optimize charge:mass ratio
|
29 |
Insufficient antibody binding affinity |
- Reduce washing stringency
- Increase antibody concentration
- Use Boster high affinity primary antibodies
|
30 |
Insufficient sample loading |
- Use more starting material
- Concentrate sample prior to loading
|