[分享] Strip Antibodies and Re-use Protein blots membrane
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Western blotting is a commonly used technique to study protein structure and function. Typically, protein samples are electrophoresed on SDS-gels and transferred to solid support (nitrocellulose or PVDF membranes) for subsequent probing with specific antibodies
Recycling of blots offers many advantages:
Effective use of samples that are available in limited amounts.
Comparison of images obtained with different antibodies in the same blot.
Confirmation of results with the same or different antibodies.
It is simply more economical and less time consuming to reuse blots.
There are many ways used for subsequent reprobing with specific antibodies after stripping, here I summerize some as follows:
One:
1) 3min in Methanol
2) 30min in PBS/5% H2O2
3) rinse in H2O for few times
4) 5 min in PBS, then ready.
Two：
1. Make Strip solution:
2% SDS
100mM beta-mercaptoethanol, or DTT
20mM Tris-Cl
1mM EDTA
2. Rinse the membrane with 100% methonal for few min.
3. Rinse 5X in H2O
4. Wash 10min in PBST
5. Transfer the membrane to strip buffer (preheated to 55-65C) for 20min,
6. Repeat step 5 once
7. Rinse 3X in PBST followed by 10min in PBST
8. Check strip efficiency (normally, it is OK, but if the expression is too high, it will be very hard to clean up).
Three：
Stripping of the membrane
• PVDF membranes may be stripped. Nitrocellulose generally does not strip well.
• Wash membrane for 30 minutes in 25 mM glycine-HCl pH 2.0 + 1% SDS with gentle shaking at room temperature.
• Rinse in PBS for 10 minutes.
• Quickly scan membrane at low resolution to determine if stripping
was successful. Repeat glycine/SDS wash and PBS rinse if necessary.
Four：
Rinse blot off with 0.05% Tween 20 in PBS.
Put blot into Kapak bag cut to slightly bigger size than blot.
Add about 5 to 10 ml Stripping buffer.
Remove as much air as possible and seal bag.
Immerse into 80°C water bath and incubate for 20 min.
Rinse blot off with 0.05% Tween 20 in PBS.
Block for about 1 hr with 5% BSA/Tween 20, or overnight with 3% BSA/Tween 20.
Five：
Stripping buffer: 0.5 L (sterile filter solution and keep at 4°C)
0.2 M Glycine, pH 2.5
0.05% Tween 20
Six：
50mM tris pH6.8, 100mM b-mercapto and 2% SDS. Leave on for 30 mins at 50C and then wash off twice with TBS-T
I've probed the same blots for different antibodies, tubulin after using a phospho antibody.
Seven：
0.5M Acetic acid
0.5M NaCl
Eight：
After your first probing，Keep the PVDF membrane wet.
1. Wash it in ddWater for 10 min
2. Wash it in 0.2 N NaOH for 10 min
3. Wash it in ddWater for anothor 10 min
4. Go to your blocking procedure
p.s. I like method Two.
Recycling of blots offers many advantages:
Effective use of samples that are available in limited amounts.
Comparison of images obtained with different antibodies in the same blot.
Confirmation of results with the same or different antibodies.
It is simply more economical and less time consuming to reuse blots.
There are many ways used for subsequent reprobing with specific antibodies after stripping, here I summerize some as follows:
One:
1) 3min in Methanol
2) 30min in PBS/5% H2O2
3) rinse in H2O for few times
4) 5 min in PBS, then ready.
Two：
1. Make Strip solution:
2% SDS
100mM beta-mercaptoethanol, or DTT
20mM Tris-Cl
1mM EDTA
2. Rinse the membrane with 100% methonal for few min.
3. Rinse 5X in H2O
4. Wash 10min in PBST
5. Transfer the membrane to strip buffer (preheated to 55-65C) for 20min,
6. Repeat step 5 once
7. Rinse 3X in PBST followed by 10min in PBST
8. Check strip efficiency (normally, it is OK, but if the expression is too high, it will be very hard to clean up).
Three：
Stripping of the membrane
• PVDF membranes may be stripped. Nitrocellulose generally does not strip well.
• Wash membrane for 30 minutes in 25 mM glycine-HCl pH 2.0 + 1% SDS with gentle shaking at room temperature.
• Rinse in PBS for 10 minutes.
• Quickly scan membrane at low resolution to determine if stripping
was successful. Repeat glycine/SDS wash and PBS rinse if necessary.
Four：
Rinse blot off with 0.05% Tween 20 in PBS.
Put blot into Kapak bag cut to slightly bigger size than blot.
Add about 5 to 10 ml Stripping buffer.
Remove as much air as possible and seal bag.
Immerse into 80°C water bath and incubate for 20 min.
Rinse blot off with 0.05% Tween 20 in PBS.
Block for about 1 hr with 5% BSA/Tween 20, or overnight with 3% BSA/Tween 20.
Five：
Stripping buffer: 0.5 L (sterile filter solution and keep at 4°C)
0.2 M Glycine, pH 2.5
0.05% Tween 20
Six：
50mM tris pH6.8, 100mM b-mercapto and 2% SDS. Leave on for 30 mins at 50C and then wash off twice with TBS-T
I've probed the same blots for different antibodies, tubulin after using a phospho antibody.
Seven：
0.5M Acetic acid
0.5M NaCl
Eight：
After your first probing，Keep the PVDF membrane wet.
1. Wash it in ddWater for 10 min
2. Wash it in 0.2 N NaOH for 10 min
3. Wash it in ddWater for anothor 10 min
4. Go to your blocking procedure
p.s. I like method Two.
