Search
Search
View additional product information for WesternBreeze™ Chromogenic Kit, anti-rabbit - FAQs (WB7105)
8 product FAQs found
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.
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.
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.
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.
Yes, you may purchase them as standalone products using the Cat. Nos. listed below:
- Cat. No. WB7003 (Antibody Wash)
- Cat. No. WB7001 (Blocker/Diluent A)
- Cat. No. WB7002 (Blocker/Diluent B)
- Cat. No. WB7050 (Combo pack containing Blocker/Diluents A & B)
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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 Chromogenic 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 Chromogenic 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 Chromogenic 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 Chromogenic 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 Chromogenic Immunodetection protocol.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Yes, you can purchase it as a standalone (Cat. No. WP20001). It will give a purple chromogenic precipitate.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
We offer the following three types of WesternBreeze Chromogenic Detection Kits:
- WesternBreeze Chromogenic Detection Kit: Anti-Mouse, Cat. No. WB7103
- WesternBreeze Chromogenic Detection Kit: Anti-Rabbit, Cat. No. WB7105
- WesternBreeze Chromogenic Detection Kit: Anti-Goat, Cat. No. WB7107
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.