Professional Documents
Culture Documents
GENERAL INFORMATION
GENERAL INFORMATION
Product Description
Western blots are used to detect specific proteins that have been separated from one another
according to their size by gel electrophoresis. Detection involves a transfer procedure followed by
probing the blot membrane with a primary antibody that recognizes specific amino acids on your
protein of interest. A secondary antibody conjugated with peroxidase is used to bind with the
primary antibody. To detect this, a chemiluminescent substrate is added to react with the antibody
complex, revealing the location and relative amount of protein.
Procedure Overview
Bioo Scientific’s MaxDiscovery™ Akt Western Blot Kit is a complete kit that contains all necessary
instructions and reagents to perform a western after transfer is complete in a clean and
user-friendly manner. Akt, also known as protein kinase B plays an essential role in cell
proliferation, cell survival, cell migration and the insulin signaling pathway. The monoclonal
antibody to PKB/Akt recognizes the Human Protein Kinase B (PKB/Akt), C terminal. There are
concentrated buffers that will require dilution before starting and can be made prior to the actual
western blot procedure.
Methanol
Rocking or gyrating platform
Small tray (Size will determine how much buffer is necessary to cover the membrane)
Ultrapure water
PVDF membrane (Bio-Rad, cat# 162-0177)
X-ray film or camera-based imaging system
The recommended membrane is PVDF for this kit. Before transfer, immerse your PVDF membrane
in methanol for 30 seconds before equilibrating it in transfer buffer for 15 minutes.
The usual “sandwich” layer orientation for transfer is:
It is highly recommended to keep the transfer as cold as possible to prevent overheating of the
buffer and membrane.
Reagent Preparation
Stock Buffer (1 Liter) 1 mini gel / 1 large gel
1 Mini: Mix 20 mL of 10X Wash Solution A with
380 mL of ultrapure water
20X Wash Solution A 1X Wash Solution A
1 Large: Mix 30mL of 10X Wash Solution A with
570 mL of ultrapure water
1 Mini: Mix 15 mL of 10X Wash Solution B with
135 mL of ultrapure water
10X Wash Solution B 1X Wash Solution B
1 Large: Mix 20 mL of 10X Wash Solution B with
180 mL of ultrapure water
1 Mini or 1 Large gel: Mix 40 mL of 2X Antibody
2X Antibody Diluent 1X Antibody Diluent
Diluent and 40 mL of 1X Wash Solution B
1. Disassemble the transfer set up described above, remove the membrane, and immediately
transfer to a tray filled with 15 mL (mini) or 25 mL (large) 1X Wash Buffer A.
2. At room temperature (20 – 25°C / 68 – 77°F), perform 2, 30 second washes with 15 mL (mini)
or 25 mL (large) 1X Wash Buffer A.
3. Perform 1, 30 second wash with 15 mL (mini) or 25 mL (large) 1X Wash Buffer B.
4. Add 40 mL of Blocking buffer to the membrane and incubate at 4° C for 2 hours with constant
rocking.
*Optional – Blocking can be done overnight at 4° C.
5. At room temperature (20 – 25°C / 68 – 77°F), perform 2, 30 second washes with 15 mL (mini)
or 25 mL (large) 1X Wash buffer A.
6. Perform 1, 30 second wash with 15 mL (mini) or 25 mL (large) 1X Wash Buffer B.
7. Make a 1:8000 antibody dilution using Akt primary antibody and 40 mL of antibody diluent.
8. Add above diluted primary antibody to the membrane and incubate at 4° C for 90 minutes with
constant rocking.
9. At room temperature (20 – 25°C / 68 – 77°F), perform 1, 30 second wash with 15 mL (mini) or
25 mL (large) 1X Wash Buffer A followed by 3, 15 minute washes with 40 mL (mini) or 60 mL
(large) 1X Wash Buffer A with constant rocking.
10. Perform 1, 30 second wash with 15 mL (mini) or 25 mL (large) 1X Wash Buffer B.
11. Make a 1:5000 antibody dilution using the secondary anti-mouse peroxidase and 40 mL of
Antibody Diluent.
12. Add the secondary antibody to the membrane and incubate at 4° C for 60 minutes with
constant rocking.
13. At room temperature (20 – 25°C / 68 – 77°F), perform 1, 30 second wash with 15 mL (mini) or
25 mL (large) 1X Wash Buffer A followed by 3, 15 minute washes with 40 mL (mini) or 60 mL
(large) 1X Wash Buffer A with constant rocking.
14. Perform 1, 30 second wash with 15 mL (mini) or 25 mL (large) 1X Wash Buffer B followed by 1,
15 minute wash with 40 mL (mini) or 60 mL (large) 1X Wash Buffer B at room temperature (20
– 25°C / 68 – 77°F), with constant rocking.
15. Place chemiluminescent substrates at room temperature (20 – 25°C / 68 – 77°F) 15 minutes
prior to use. Mix 400 µl of chemiluminescent substrate A with 400 µl of chemiluminescent
substrate B.
16. At room temperature (20 – 25°C / 68 – 77°F), incubate the membrane with chemiluminescent
substrate for 5 minutes prior to exposure. Be sure to place the substrate directly on the side of
the membrane that faced the gel.
17. Membrane can be visualized using X-Ray film or digital imaging.
TROUBLESHOOTING TROUBLESHOOTING
Tired of Troubleshooting your Western Blot? Let BIOO perform this service for you. Contact us at
services@biooscientific.com for details.
No Signal
Possible Causes Recommended Action
Insufficient amount of primary Lower the dilution factor or perform dot blots to optimize the
antibody antibody dilution.
Primary and secondary antibodies are Be sure to check both antibodies are compatible. If primary
incompatible antibody is mouse, secondary antibody must be anti-mouse.
BSA is a protein based blocking buffer and should not interfere with
Over-blocking or wrong blocking most westerns. However, blocking buffers can be optimized by
buffer. trying other blocking buffers such as 5% nonfat dry milk, 1%
casein, or non-mammalian protein buffer.
Transfer may have been too hot. Be sure to use cold buffers and
leave the transfer chamber in 4° C instead of room temperature.
Poor Transfer Be sure to equilibrate membrane, gel, and filter papers in transfer
buffer. PVDF must be soaked in methanol and then equilibrated in
transfer buffer first.
Be sure to use at least 10-50 µg of protein in each minigel well. If
Insufficient amount of antigen the antigen is expressed in small amounts, increase the amount of
protein.
Make fresh mixes of substrate A and substrate B for each western.
Substrate is old or inactive Do not mix in advance. Once mixed, substrate is good for only 8
hours in room temperature.
Do not over wash the membrane. If there are lag times, leave the
Excessive Washing
membrane in Wash Buffer B.
High Background
Possible Causes Recommended Action
Poor Blocking Be sure to block for at least 2 hours or overnight at 4° C only.
Increase the dilution factor for primary antibody or secondary
Antibody dilution may be too high
antibody. Perform dot blots to optimize the antibody dilutions.
Secondary or primary antibodies may be binding to the blocking
Antibody may be reacting to buffers buffer. Switch to other types of buffers such as nonfat dry milk,
casein, or non-protein based.
If the membrane ever dries out, be sure to soak the PVDF
Membrane dried out
membrane in methanol and then in Wash Buffer B.
Nonspecific Bands
Possible Causes Recommended Action
Too much HRP Use less HRP-conjugate by dilution.
Equilibrate the gel in transfer buffer before assembling the transfer.
SDS caused nonspecific binding
Do not use SDS in any of the procedures.
BIOO will include specific concentration and dilution information for antibodies purchased for
Westerns from us.
If you need further technical assistance, you may contact a representative at BIOO:
techsupport5@biooscientific.com.
1.888.208.2246
BIOO makes no warranty of any kind, either expressed or implied, except that the materials from which its products are
made are of standard quality. There is no warranty of merchantability of this product, or of the fitness of the product for
any purpose. BIOO shall not be liable for any damages, including special or consequential damage, or expense arising
directly or indirectly from the use of this product.
Bioo Scientific Corporation Made in USA
3913 Todd Lane Suite 312 BIOO Research Products Group
Austin, TX 78744 USA info@biooscientific.com
Tel: 1.888.208.2246 techsupport5@biooscientific.com
Fax: (512) 707-8122 www.biooscientific.com