Immunoprecipitation Kit (Protein A Agarose Gel)
Immunoprecipitation (IP) or co-immunoprecipitation (Co-IP) is the study of proteins or protein-protein interactions. PPIs) are used to separate the target protein from complex samples by using specific antibodies and antibody-binding media (such as Protein A/G agarose gels, etc.), or directly using media conjugated to specific antibodies (such as agarose gels or magnetic beads), which can be used for Western blot detection or mass spectrometry analysis.
Protein A is a cell wall surface protein found in Staphylococcus aureus with a molecular weight of 42 kDa, which specifically binds to the Fc region of mammalian immunoglobulin (Ig) and also to the Fab region of the human VH3 family. Protein A specifically binds to antibodies such as human IgG1, IgG2, IgG4, mouse IgG2a, IgG2b, and rabbit IgG, and each Protein A molecule can bind up to 5 IgG molecules. The binding capacity of common immunoglobulin subclasses and the total binding capacity of different species are shown in the table below (Table 1).
This kit contains Protein A agarose gel and other necessary reagents for immunoprecipitation, and with the specific antibodies provided by the user, it makes immunoprecipitation (IP, also known as pull-down) or co-immunoprecipitation (Co-IP) experiments simpler, more convenient and efficient, and the immunoprecipitation products can be used for the detection of the target protein or its protein complexes and other experiments. (See Table 2 for specific product parameters).
|
Species |
Subclass |
Protein A binding |
Protein G binding |
|
Human |
lgA |
++ |
- |
|
lgD |
++ |
- |
|
|
IgE |
++ |
- |
|
|
lgG1 |
++++ |
++++ |
|
|
lgG2 |
++++ |
++++ |
|
|
IgG3 |
- |
++++ |
|
|
lgG4 |
++++ |
++++ |
|
|
lgM |
++ |
- |
|
|
Mouse |
IgG1 |
+ |
++++ |
|
lgG2a |
++++ |
++++ |
|
|
lgG2b |
+++ |
+++ |
|
|
lgG3 |
++ |
+++ |
|
|
lgM |
+/- |
- |
|
|
Rat |
lgG1 |
- |
+ |
|
lgG2a |
- |
++++ |
|
|
lgG2b |
- |
++ |
|
|
lgG3 |
+ |
++ |
|
|
Avian egg yolk |
IgY |
- |
- |
|
Cow |
|
++ |
++++ |
|
Dog |
|
++ |
+ |
|
Goat |
|
- |
++ |
|
Guinea Pig |
lgG1 |
++++ |
++ |
|
lgG2 |
++++ |
++ |
|
|
Hamster |
|
+ |
++ |
|
Horse |
|
++ |
++++ |
|
Koala |
|
- |
+ |
|
Llama |
|
- |
+ |
|
Monkey (rhesus) |
|
++++ |
++++ |
|
Pig |
|
+++ |
+++ |
|
Rabbit |
|
++++ |
+++ |
|
Sheep |
|
+/- |
++ |
Table 1 Affinity data for Protein A for different sources and subtypes of IgG. ++++ = Strong Binding; ++~+++ = Medium Binding; + = Weak Binding; +/- = Weak or No Binding; - = No Binding.
|
Characteristics |
Description |
|
Product content |
Store in 20% ethanol with a glue-to-suspension ratio of approximately 75% |
|
Bead structure |
4% cross-linked agarose |
|
Average beads size |
90 μm |
|
Coupled protein |
Recombinant Protein A (E. coli) |
|
M. W. of protein |
14 kDa Protein A |
|
Ligand concentration |
6 mg rProtein A per mL settled gel |
|
Binding capacity |
~35 mg human IgG per ml settled gel |
|
Elution method |
Elution with acid, competing peptide or SDS-PAGE loading buffer |
|
Operating temperature |
2-40℃ |
|
pH stability* |
3-10 |
|
Solvent resistant |
6 M guanidine hydrochloride, 8 M urea, 2% benzyl alcohol**, 75% ethanol**, 1 mM NaOH (pH 11)., 0.1 M sodium citrate/HCl (pH 3). |
|
Application |
Suitable for IP, Co-IP, Protein purification |
|
Storage |
4°C |
|
*After 7 days of storage at 40°C, pH3-10, the physicochemical properties, and functions of agarose showed no significant changes. ** v/v, volume ratio |
|
Table 2 The main related indicators of Protein A Agarose Gel.
| Components | K4621-20 T |
| Cell Lysis Buffer | 50 mL |
| Protease Inhibitor Cocktail (EDTA-Free,100X in DMSO) | 0.5 mL |
| 10X TBS | 10 mL |
| Neutralization Buffer | 200 μL |
| Acid Elution Buffer | 2 mL |
| Protein A Agarose Gel | 400 μL |
| 5X Protein Loading Buffer (Reducing) | 600 μL |
Store Protease Inhibitor Cocktail (EDTA-Free,100X in DMSO) and 5X Protein Loading Buffer (Reducing) at -20°C and the rest of components at 4°C for 12 months. | |