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OneMinute
Western Blot Stripping Buffer
In
ONE minute, your membrane is ready for reprobing |
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For saving time and precious samples, researchers often strip off the first set of primary and secondary antibodies after performing Western Blot detection in order to detect different proteins on the same membrane. However, they usually suffer either significant loss of target proteins from stripping the blot using harsh stripping buffer, or incomplete removal of the antibodies from using mild stripping
buffer. OneMinute Western Blot Stripping Buffer was developed to help you effectively strip off high-affinity antibodies without removal of transferred proteins on the membrane.
OneMinute Western Blot Stripping Buffer, a revolutionary stripping buffer, immediately makes your membrane ready for multiple re-probes in
ONE minute. It provides a faster and easier way to strip off high-affinity primary and secondary antibodies on the membrane. It reduces incubation times while stripping at room temperature, and re-blocking the blots usually is not necessary.
It spends only 30 seconds for stripping and 3x 10 seconds for washing.
OneMinute Western Blot Stripping Buffer is harsh for antibodies, but gentle to transferred proteins on the membrane. It effectively strips off primary and secondary antibodies without removal of transferred proteins, which allows the use of a single membrane for up to 10 times re-probes. In contrast, traditional stripping condition is so harsh that loss of transferred proteins is inevitable and the blots can hardly be re-probed for more than 2~3 times.
OneMinute Western Blot Stripping Buffer
is effective with both nitrocellulose and PVDF membranes, and various antibody conjugates (HRP, AP, etc.).
It is conveniently stored at room temperature and used without dilution.
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stripping your blots? Getting tired of tedious procedure
of stripping? OneMinute Western Blot Stripping Buffer
is
your best choice.
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Highlights:
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1.
Faster and Easier: less than 1 minute
2.
Multiple times of re-probes (~10 times)
3.
Re-blocking is usually not necessary
4.
Perform at room temperature
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Protocols:
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1.
Immerse membrane in OneMinute Western Blot Stripping Buffer
for 30 sec.
2. Wash the blot with Washing Buffer (TBST or
PBST) 3 x 10 seconds*.
*The
blot is ready for re-probing. (Note: Reblocking
membrane is usually not necessary.) |
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OneMinute: 30sec at RT, washing 3x 10 sec, No re-blocking.
Competitor: 15min at 50℃, washing 3x 10min, re-blocking 1 hour, washing 3x 15min |
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Figure 1.
Re-probing with different antibodies. 5% milk
blocked nitrocellulose Western blots of 293 cell lysate
were probed with Rabbit-anti-Human JNK1 (Protein1, Santa
Cruz, sc-747) at 1:200 dilution, HRP-Goat-anti-Rabbit
(Amersham) at 1:10,000 dilution. The signal was detected
using ECL substrate (Panel A). Blots were stripped with
either 2ml of OneMinute Stripping Buffer (30
sec, RT) or 10ml of competitor stripping buffer
(15min, 50℃) (Panel B). OneMinute
Buffer treated blot was washed 3x 10sec with TBST, and
performed re-probing directly without re-blocking.
Competitor stripping buffer treated blot was washed 3x
10min, blocked by 5% milk for 1 hour, and washed 3x 15min
with TBST. Blots were re-probed with Mouse-anti-Human
RIPK1 (Protein2, BD, 610458) at 1:200 dilution, HRP-Goat-anti-Mouse (Amersham) at
1:2,000 dilution and detected with ECL substrate (Panel
C).
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Figure
2. Reprobing with different antibodies from same host.
5% milk
nitrocellulose Western blots of HeLa cell lysate proteins were probed with
Mouse-anti-Human RIPK1 (protein
1, BD, 610458) at 1:200 dilution, HRP- Goat-anti-Mouse (Amersham) at
1:2,000 dilution. The signal was detected using ECL substrate (Panel A). Blots were stripped with
2ml of OneMinute Stripping Buffer (30sec,
RT),
or 10ml of competitor stripping buffer (P's R product) for
either 30 sec or 15min at RT (Panel B). OneMinute
Buffer treated blot was washed 3x 10 sec with TBST, and
directly re-probed without re-blocking. Competitor stripping buffer treated blot was washed 3x 10min, blocked with 5% milk for 1hour, and washed 3x 15min with TBST. Blots were re-probed with
Mouse-anti-Human IKKgamma (protein
2, BD, 559675) at 1:200 dilution,
HRP-Goat-anti-Mouse (Amersham) at 1:2,000 dilution and detected with ECL substrate (Panel
C). The removal of the primary
antibody against Protein 1 was also evaluated by using
same secondary antibodies (Anti-Mouse). 15min stripping
with competitor P's R product completely removed
secondary antibody (right lane in Panel B) but failed to
completely remove primary antibody (right lane in Panel
C). |
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Figure
3. OneMinute Stripping Buffer Completely removes high affinity antibodies (both
primary and secondary). 5% milk blocked Nitrocellulose
Western blots of 293 cell lysate were probed with
Mouse-anti-Human Actin
(Sigma, A-5441) at 1:5000 dilution, HRP-Goat-anti-Mouse
(Amersham) at 1: 2,000 diluiton. The signal was detected using ECL substrate (Panel A). Blots were stripped with either
2ml of OneMinute Stripping Buffer ( 1min,
RT), or 10ml of competitor stripping buffer (P's R
product, 15min, RT)
(Panel B). To further test the removal of primary
antibody, the stripped blots were re-probed with identical
secondary antibody and detected with ECL substrate (Panel
C).
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Figure
4. Re-probed for different proteins on PVDF membranes.
5% milk blocked PVDF Western blots of
HeLa cell lysate were probed with
Mouse-anti-Human RIPK1
(protein1, BD, 610458) at 1:200 dilution, HRP-Goat-anti-Mouse
(Amersham) at 1:2,000 dilution. The signal was detected using ECL substrate (Panel A). Blots were stripped with either
2ml of OneMinute Stripping Buffer(30sec,
RT) or 10ml of competitor stripping buffer
(P's R product, 30sec, RT) (Panel
B). OneMinute Buffer treated blot was washed 3x 10
sec with TBST, and directly re-probed without re-blocking. Competitor stripping buffer treated blot was washed 3x 10min, blocked with 5% milk for 1hour, and washed 3x 15min with TBST.
Blots were then re-probed with Mouse-anti-Human IKKgamma
(protein2, BD, 559675) at 1:200 dilution,
HRP-Goat-anti-Mouse (Amersham) at 1:2,000 dilution and detected with ECL substrate (Panel C).
The removal of the primary
antibody against Protein 1 was also evaluated by using
same secondary antibodies (Anti-Mouse).
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OneMinute: 10sec at RT, washing 3x 10 sec, No re-blocking.
Competitor: 15min at 50℃, washing 3x 10min, re-blocking 1 hour, washing 3x 15min |
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Figure
5.
Stripping by OneMinute Buffer does not damage transferred proteins
on the membrane. 5% milk blocked nitrocellulose
Western blots of 293 cell lysate were stained with Ponceau
S red (Panel A). Blots were stripped using either 2ml of OneMinute
Stripping Buffer (30 sec, RT) or 10ml of competitor
stripping buffer (15min, 50℃), and then stained
with Ponceau S red (Panel B). OneMintue
Buffer treated blot was washed 3x 10 seconds with TBST,
and performed re-bloting directly without re-blocking.
Competitor stripping buffer treated blot was washed 3x
10min, blocked with 5% milk for 1 hour, and washed 3x
15min with TBST. Blots were re-probed with
Rabbit-anti-Human JNK1 (Santa Cruz, sc-747) at 1:200
dilution, HRP-Goat-anti-Rabbit (Amersham) at 1:10,000
dilution. The signal was detected using ECL substrate
(Panel C).
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OneMIntue: strip 10 times.
Competitor: strip 1 time |
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Figure
6. Multiple times of stripping with OneMinute stripping
buffer remains transferred proteins on membranes.
5% milk blocked nitrocellulose Western blots of 293
cell lysate were probed with Rabbit-anti-Human JNK1 (Santa
Cruz, sc-747) at 1:200 dilution, HRP-Goat-anti-Rabbit (Amersham)
at 1:10,000 dilution. The signal was detected using ECL
substrate (Panel A). Blots were stripped with either 2ml
of OneMinute Stripping Buffer (30 sec, RT)
for 10 times or 10ml of competitor stripping buffer
(15min, 50℃) for 1 time. OneMinute
Buffer treated blot was washed 3x 10 sec with TBST, and
directly re-probed without re-blocking. Competitor
stripping buffer treated blot was washed 3x 10min,
re-blocked with 5% milk for 1hour, and washed 3x 15min
with TBST. Blots were re-probed with the same primary and
secondary antibodies and detected with ECL substrate
(Panel B). A fresh identical blot was probed as a control
(left in Panel B) to evaluate the loss of JNK on
membranes.
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OneMinute:
30sec at RT, washing 3x 10 sec, no re-blocking.
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Figure
7. Detect phosphorylated and non-phosphorylated forms of the same protein.
5% milk blocked nitrocellulose Western blots of 293 cell
lysate stimulated with or without Sorbitol (0.6M, 20min)
were probed with Rabbit-anti-Human phospoho-JNK1/2
(T183/Y185) (Cell Signaling Technology, #9251) at 1:1000
dilution, HRP- Goat-anti-Rabbit (Amersham) at 1:10,000
dilution. The signal was detected using ECL substrate
(Panel A). Blots were stripped using 2ml of OneMinute
Stripping buffer (30 sec, RT), and washed 3x 10 seconds
with TBST (Panel B). Blots were re-probed with Mouse
anti-Human JNK1/2 (BD, #554285) at 1:1000 dilution, HRP-Goat-anti-Mouse
(Amersham) at 1:2,000 dilution and
detected with ECL substrate (Panel C).
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Ordering
Information |
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Click
here to download the product manual |
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