Remove the primary and secondary Remove the primary and secondary

Stripping

Stripping

Purpose Purpose

Remove the primary and secondary Remove the primary and secondary

antibodies from a western blot membrane.

antibodies from a western blot membrane.

Investigate more than one protein on the Investigate more than one protein on the

same blot. (Interested protein and a loading same blot. (Interested protein and a loading

control) control)

The advantage of saving samples, materials, The advantage of saving samples, materials, and time.

and time.

Mild stripping Mild stripping

stripping Buffer, 1 liter stripping Buffer, 1 liter

glycine

glycine 15 g 15 g SDS 1 g SDS 1 g

Tween

Tween 20 10 ml 20 10 ml Adjust pH to 2.2

Adjust pH to 2.2

Bring volume up to 1 L with ultra pure water

Bring volume up to 1 L with ultra pure water . .

Mild stripping Mild stripping

1. 1. Use a volume that will cover the Use a volume that will cover the

membrane. Incubate at room temperature membrane. Incubate at room temperature

for 5

for 5 - - 10 minutes. 10 minutes.

2. 2. Discard buffer. Discard buffer.

3. 3. 5 5 - - 10 minutes fresh stripping buffer. 10 minutes fresh stripping buffer.

4. 4. Discard buffer. Discard buffer.

5. 5. 10 minutes PBS and Discard (2 times) 10 minutes PBS and Discard (2 times)

Harsh stripping Harsh stripping

Buffer, 0.1

Buffer, 0.1 litre litre

SDS 10% 20 ml SDS 10% 20 ml Tris Tris - - HCl HCl , pH 6.8, 0.5 M 12.5 ml , pH 6.8, 0.5 M 12.5 ml

ultra pure water 67.5 ml ultra pure water 67.5 ml ß ß - - mercaptoethanol mercaptoethanol 0.8 ml 0.8 ml

Prepare buffer and strip membranes under a Prepare buffer and strip membranes under a fumehood

fumehood. .

Harsh stripping Harsh stripping

1. 1. Warm the buffer to 50 Warm the buffer to 50 ° ° C. C.

2. 2. Add the buffer to a small plastic box which Add the buffer to a small plastic box which has a tight lid. Use a volume that will cover has a tight lid. Use a volume that will cover

the membrane.

the membrane.

3. 3. Add the membrane. Incubate at 50 Add the membrane. Incubate at 50 ° ° C for C for up to 45 minutes with some agitation.

up to 45 minutes with some agitation.

4. 4. Dispose buffer. Dispose buffer.

5. 5. Rinse the membrane under running water Rinse the membrane under running water tap for 1

tap for 1 - - 2 hours. 2 hours.

Checking the removal of Checking the removal of

HRP label HRP label

Incubate the membrane with fresh Incubate the membrane with fresh chemiluminescence

chemiluminescence reagents and expose to reagents and expose to film.

film.

If the signal is detected with a 5 minute If the signal is detected with a 5 minute exposure or not.

exposure or not.

Checking the removal of Checking the removal of

primary antibody primary antibody

1. 1. Incubate the membrane with the fresh Incubate the membrane with the fresh HRP labeled secondary antibody.

HRP labeled secondary antibody.

2. 2. Followed by a wash in wash buffer. Followed by a wash in wash buffer.

Incubate the membrane with fresh Incubate the membrane with fresh

chemiluminescence

chemiluminescence reagents and expose reagents and expose to film.

to film.

3. 3. If the signal is detected with a 5 minute If the signal is detected with a 5 minute exposure or not.

exposure or not.