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Can the labeling reaction tolerate the presence of BSA or gelatin?

Labeling IgG free of BSA or gelatin stabilizer gives the best results. However, IgG containing BSA or gelatin can be labeled with good results using a larger sized kit and our modified Mix-n-Stain protocol. See the Mix-n-Stain Kit Selection Guide below to select the appropriate kit size and protocol.

Antibody formulation Kit and protocol selection Notes/Examples
Purified IgG containing:

• Sodium azide

• Less than 20 mM Tris

• Less than 10% glycerol

1. Choose the kit size based on the amount of IgG you wish to label

2. Use the standard labeling protocol

If the amount of antibody you wish to label falls between two kits sizes, we recommend using the smaller kit size. For example, if you wish to label 20 ug IgG, choose the 5-20 ug-sized kit.
Purified IgG containing:

• More than 20 mM Tris

• More than 10% glycerol

• Glycine

• Less than 0.5 ug/uL IgG

1. Choose the kit size based on the amount of IgG you wish to label

2. Perform ultrafiltration using the spin vial provided in the kit

3. Use the standard labeling protocol

If the amount of antibody you wish to label falls between two kits sizes, we recommend using the smaller kit size. For example, if you wish to label 20 ug IgG, choose the 5-20 ug-sized kit.
Purified IgG containing:

• Less than 4:1 BSA:IgG by weight

• Less than 4:1 gelatin:IgG by weight

1. Choose the kit size based on the amount of IgG you wish to label

2. Use the standard labeling protocol

For example, if you wish to label 5 ug IgG in 5 uL PBS contain­ing 0.1% BSA. The BSA:IgG ratio by weight is 5 ug BSA:5 ug IgG or 1:1. Select a 5-20 ug-sized kit and follow the standard protocol.
Purified IgG containing:

• More than 4:1 BSA:IgG by weight

• More than 4:1 gelatin:IgG by weight

1. Choose the kit size based on the total amount of protein (IgG + BSA/gelatin) in the volume of antibody solution you wish to label

2. Use the modified labeling protocol

For example, if you wish to label 5 ug IgG in 5 uL PBS containing 1% BSA. The BSA:IgG ratio by weight is 50 ug BSA: 5 ug IgG or 10/1. Select a 50-100 ug-sized kit based on 55 ug of total protein in the labeling reaction and follow the modified labeling protocol. If the total protein amount falls between two kit sizes, you may get better results with the larger kit size.
IgG in ascites fluid 1. Determine the concentration of protein in the ascites fluid

2. Choose the kit size based on the total amount of protein in the volume of ascites fluid you wish to label

3. Use the modified labeling protocol

For example, if you wish to label 10 uL ascites fluid containing 70 ug total protein. Select a 50-100 ug-sized kit based on 70 ug of total protein, and follow the modified labeling protocol.
Less than 5 ug purified IgG 1. Add stabilizer protein such as BSA to the antibody to bring the total amount of protein 5 ug

2. Select a 5-20 ug kit and use the standard labeling protocol

For example, if you wish to label 1 ug IgG sample. Select a 5-20 ug-sized kit. Before labeling, add 4 ug BSA to 1 ug IgG and follow the standard labeling protocol.
IgG in:

• Serum

• Hybridoma cell culture supernatant

Not compatible due to the low concentration of IgG compared to other proteins in these formats; purify IgG before labeling Use protein A/G or a commercially available IgG clean-up kit to purify IgG. Determine the concentration of IgG, then select the appropriate kit/protocol based on the amount of IgG you wish to label and the buffer formulation after purification.

Category: Mix-n-Stain™ Antibody Labeling Kits

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