WesternBreeze™ Chemiluminescent Kit, anti-mouse - FAQs

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12 product FAQs found

Why is the actual band size on a western blot different from the predicted size of the protein?

Western blotting is based on the separation of proteins by their size on a gel. However, migration of proteins through the gel matrix is also affected by other factors, which may cause the observed band size to be different from the predicted size.

Common causes are:
-Post-translational modification; for example phosphorylation and glycosylation increase the size of the protein
-Post-translation cleavage; many proteins are synthesized as precursor proteins, and then cleaved to give the active form
-Multimers, for example dimerization of a protein. This is usually prevented under reducing conditions, although strong interactions can result in the appearance of higher bands
-Splice variants; alternative splicing may result in different sized proteins being produced from the same gene
-Relative charge; the composition of amino acids (charged vs. non-charged)

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

What are the standard lysis buffers used with mammalian cells for detection of protein expression by immunoprecipitation (IP) or Western blot analysis?

The most commonly used buffer is RIPA Buffer with SDS. We offer RIPA Buffer (Cat. Nos. 89900 and 89901). We also offer the Pierce IP Lysis buffer (Cat. Nos. 87787 and 87788) as well as M-PER (Cat. Nos. 78501, 78503, and 78505).

Find additional tips, troubleshooting help, and resources within our Cell Lysis and Fractionation Support Center.

What conditions do you recommend for overnight Western transfer?

Doing an overnight Westerm transfer is not the preferred method but can be done. The power should be lowered and the buffer should be chilled or the unit should be placed in the cold room to prevent overheating. You may try an overnight transfer at 5-15 V and adjust accordingly. You may also wish to put a second membrane behind the first in order to bind any proteins that transfer through the first membrane. You can use both membranes for staining, immunoblotting, or analysis.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

After transferring onto a PVDF membrane, what is the best way to store the membrane for future probing?

We recommend air drying the PVDF membrane and placing it in an envelope, preferably on top of a supported surface to keep the membrane flat. It can be stored indefinitely at ≤80 degrees C. Right before probing, we recommend re-wetting the membrane with alcohol for a few seconds, followed by a few rinses with pure water to reduce the alcohol concentration. Then proceed as normal with the blocking step.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Which transfer buffers are recommended for peptide (N-terminal) sequencing?

Use non-glycine based buffers such as the NuPAGE Invitrogen Transfer buffer, CAPS, or 1/2X TBE transfer buffer.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

What parameters can affect Western blotting transfer results?

SDS:
-Mobility out of the gel: increases mobility as SDS imparts negative charge to a protein and maintains it in a soluble state.
-Binding to membrane: reduces binding to nitrocellulose due to decreased hydrophobicity of the protein.
-Detection: may affect antigenicity of some proteins.

Alcohol in transfer buffer (i.e., methanol up to 20%):
-Mobility out of the gel: decreased; reduces pore size of gel.
-Binding to membrane: improves binding to nitrocellulose; removes SDS from proteins resulting in improved hydrophobic interactions.

Field strength (V/cm):
-Mobility out of the gel: field strength is the driving force of elution; if too low, sample is not completely transferred out of gel.
-Binding to membrane: if too high, sample passes through membrane without binding.

Transfer buffer:
-Mobility out of the gel: decreased if high conductivity and low resistance limit V/cm due to excessive heat generation; decreased if buffer pH is close to pI of native protein.

Membrane:
-Detection: PVDF and nylon require more stringent blocking conditions than nitrocellulose.

Gel:
-Mobility out of the gel: increases with thinner gels or larger gel pore size.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

How can I reduce background bands in my Western blot?

Optimize the concentration of primary and secondary antibodies. In some cases, increasing the concentration of blocking agent (BSA or non-fat dry milk) or usiing an alternative blocking solution such as Starting Block or SuperBlock may reduce background signal. After incubation with the primary antibody, wash at least 2 times with TBST (include 0.5 M NaCl in one or more of the wash steps). Avoid Nonidet P40 or Triton X-100 in buffers because protein detection is decreased when these detergents are used.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

How can I increase the sensitivity of my Western blot?

Consider transferring to a different membrane or using a different detection method. We have observed increased sensitivity when using PVDF membranes in place of nitrocellulose. On PVDF membranes, using as little as 1 µg of total rat brain protein, PKC can be detected with alkaline phosphatase-mediated chromogenic detection in some cases using affinity-purified antibodies at a concentration of 0.5 µg/mL. Detection sensitivity can also be increased by using chemiluminescent detection, especially when using a SuperSignal West enhanced chemiluminescence subtrate (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/western-blot-detection-reagents/detection-technologies-western-blotting/chemiluminescent-western-blot-detection/supersignal-chemiluminescent-substrates.html) such as SuperSignal West Pico PLUS, SuperSignal West Dura, or SuperSignal West Femto. The secondary antibody should be used as recommend by the manufacturer and optimized as needed.

Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.

Can I purchase the Secondary Antibody solutions from the WesternBreeze Chemiluminescent Detection kits as standalone products?

The goat anti-mouse and goat anti-rabbit secondary antibody solutions AP-conjugated are available as standalone products (Cat. Nos. WP20006 and WP20007), but the rabbit anti-goat secondary antibody solution AP-conjugated is not available as a standalone product.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I purchase the Blocker/Diluent and Antibody Wash solutions from the WesternBreeze Chemiluminescent Detection kits as standalone products?

Yes, you may purchase them as standalone products using the Cat. Nos. listed below:

- Cat. No. WB7001 (Blocker/Diluent A)
- Cat. No. WB7002 (Blocker/Diluent B)
- Cat. No. WB7050 (Combo pack containing Blocker/Diluents A & B)
- Cat. No. WB7003 (Antibody Wash)

Note: Cat. Nos. WB7001 and WB7002 may be ordered by clicking the Quick Order button located at the top right of any page on our website.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Can I purchase the Chemiluminescent Substrate and Chemiluminescent Substrate Enhancer from the WesternBreeze Chemiluminescent Detection Kits as standalone products?

Yes, you can purchase them as standalone products and below are the Cat. Nos.:

- Chemiluminescent Substrate, Cat. No. WP20002
- Chemiluminescent Substrate Enhancer, Cat. No. WP20003

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

How should I prepare my membranes for WesternBreeze Chemiluminescent detection?

For western blots, where proteins are freshly transferred from SDS-PAGE gels to nitrocellulose or PVDF membranes, washing the membranes twice for 5 mins with 20 mL of pure water is recommended to partially remove gel and transfer buffer components and weakly bound proteins. The membranes are then ready for the WesternBreeze Chemiluminescent Immunodetection protocol.

Alternatively, the washed membranes may be dried on a clean piece of filter paper in open air, by a stream of slightly warm air or under an infrared lamp. Properly dried membranes can be stored in a closed container at 4 degrees C for several days depending on the antigen loaded. Water-washed and dried nitrocellulose membranes are ready for the WesternBreeze Chemiluminescent Immunodetection protocol. However, water-washed and dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 mins before proceeding to the WesternBreeze Chemiluminescent Immunodetection protocol.

For Native-PAGE western blot, a drying step, performed before any washing steps, is recommended to improve protein binding to the membrane. Once dried, nitrocellulose membranes should be washed twice with 20 mL water for 5 mins before proceeding to the WesternBreeze Chemiluminescent Immunodetection protocol. Dried PVDF membranes require a re-wetting step in methanol, followed by two 20 mL water washes for 5 minutes before proceeding to the WesternBreeze Chemiluminescent Immunodetection protocol.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.