Protein Analysis FAQ

• • Does Storing Samples at -20°C for 3 Months Affect Detection Results?

  For most biological and chemical samples, storage at -20°C for up to three months generally does not significantly impact the detection results. However, the specific effects depend on the sample type and detection method. Biological samples such as DNA, RNA, and proteins can typically be stored at -20°C for extended periods without affecting subsequent molecular biology tests like PCR, Western blotting, or ELISA. However, repeated freeze-thaw cycles may damage these molecules, so it is best to avoid ......

• • What Is the Function of Histone Acetylation, and What Role Does HAT Play in This Process?

  Histone acetylation is an epigenetic modification that plays a crucial role in regulating gene expression and DNA repair. In this process, specific enzymes transfer acetyl groups (provided by acetyl-CoA) to lysine residues on histones, thereby influencing chromatin structure and function.   Functions of Histone Acetylation 1. Chromatin Relaxation The addition of acetyl groups to histones reduces their positive charge, weakening the interaction with negatively charged DNA. This results in a more relaxe......

• • What Are the Differences Between SDS and Non-SDS Systems in Protein Gel Electrophoresis?

  When performing protein gel electrophoresis, the inclusion or exclusion of SDS (sodium dodecyl sulfate) leads to distinct differences in the following aspects:   Sample Preparation 1. With SDS Adding SDS during sample preparation imparts a uniform negative charge to protein molecules and linearizes them by disrupting their secondary and tertiary structures.   2. Without SDS Omitting SDS means proteins retain their native charges and conformations during sample preparation.   Gel Preparation 1. With SD......

• • Why Doesn't the Protein Marker Separate in SDS-PAGE Electrophoresis?

  SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is a widely used technique for protein analysis, allowing separation based on molecular weight. Protein markers serve as essential references to estimate the molecular weights of sample proteins. If the protein marker fails to separate during electrophoresis, several factors might be responsible:   1. Inappropriate Electrophoresis Conditions Settings such as voltage, current, and duration are crucial. Incorrect parameters can hinder ......

• • What Causes Baseline Drift During HPLC Sample Injection?

  To determine the amount of a 100-unit enzyme required for protein digestion, follow these steps:   1. Determine the Total Amount of Protein For example, if you have 1 milligram (mg) of protein.   2. Calculate the Required Enzyme Amount For Asp-N: If the recommended ratio is 50:1, the required amount of Asp-N is 1 mg / 50 = 0.02 mg.   For Glu-C: If the recommended ratio is 20:1, the required amount of Glu-C is 1 mg / 20 = 0.05 mg.   3. Convert Enzyme Amount to Units Determine how many units are present......

• • Why Is the Protein Marker Not Visible on the Membrane After Western Blot Transfer?

  This issue pertains to a common occurrence in Western blot experiments. When the protein marker (molecular weight marker) is not visible on the membrane, several factors may be responsible. The following four points are potential primary causes:   1. Selection of Protein Marker The choice of marker should be based on the molecular weight of the target protein. If the marker's molecular weight is too close to that of the target protein, or if it falls outside the target protein's range, the marker may ......

• • How to Calculate the Amount of 100 Units of Enzyme Needed for Protein Digestion?

  To determine the amount of a 100-unit enzyme required for protein digestion, follow these steps:   1. Determine the Total Amount of Protein For example, if you have 1 milligram (mg) of protein.   2. Calculate the Required Enzyme Amount For Asp-N: If the recommended ratio is 50:1, the required amount of Asp-N is 1 mg / 50 = 0.02 mg.   For Glu-C: If the recommended ratio is 20:1, the required amount of Glu-C is 1 mg / 20 = 0.05 mg.   3. Convert Enzyme Amount to Units Determine how many units are present......

• • Why Does the WB Band Appear Diffuse, Resembling Protein Separation from the Loading Control?

  When Western blot (WB) bands appear diffuse or abnormally wide, several factors may contribute to this phenomenon, giving the impression of protein separation from the loading control. The potential causes include:   1. Excessive Protein Loading Overloading the sample can cause protein diffusion within the gel, leading to broad and indistinct bands. This effect is particularly pronounced when the sample concentration exceeds the gel’s resolving capacity.   To improve band sharpness, it is essential to......

• • How to Interpret the Electrophoresis Results of SDS-PAGE for Target Protein Detection?

  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used technique for protein separation. This method relies on sodium dodecyl sulfate (SDS) to denature proteins and confer a uniform negative charge, facilitating their separation in a polyacrylamide gel. Since SDS eliminates charge-based differences, protein migration is primarily dictated by molecular weight rather than the protein’s intrinsic charge or conformation.   Interpreting SDS-PAGE Electrophoresis Results Follow......

• • How Can Protein Bands Be Analyzed in SDS-PAGE Purity Assessment?

  Band Resolution and Integrity The clarity and integrity of protein bands should be carefully examined. Diffuse or smeared bands may indicate protein aggregation, degradation, or the presence of impurities.   Number of Bands The presence of multiple bands within a single lane suggests the coexistence of multiple protein species, such as contaminants or degradation products. If the sample is expected to contain a single purified protein, only one distinct band should be observed.   Background Cleanlines......