SHARDA UNIVERSITY

GREATOR NOIDA KNOWLEDGE PARK III,

UTTARPRADESH

BOTONY ASSIGNMENT
PULSE CHASE EXPERIMENT

Submitted To: Submitted By:

Ms. Anjali Malik Robin Solanki
Department of Forensic Science 2019004698
Sharda University BSc. Forensic Science
Uttar Pradesh Sharda University

PULSE CHASE EXPERIMENT-

Introduction

The pulse-chase analysis is a powerful technique to study the synthesis, processing, and transport
of proteins. The pulse-chase analysis is widely used in biochemistry and molecular biology for
the examination of the cellular process by exposing the cells to a labeled compound (pulse). The
labeled compound is introduced into the molecule or pathway being studied. In the chase phase,
an unlabeled form of that compound replaces the labeled compound. The reaction is monitored
to note the time taken by the unlabeled form of the compound to replace the labeled form. These
experiments allow the assessment and observation of the evolution of a biological process over
time.

Principle

Pulse-chase analysis is a specialized form of metabolic labeling which employs radioactive

amino acids for observing cellular events over time. The radioactive amino acids are added in the
cells for a few minutes (the “pulse”), washed away, and then the cells are exposed to
nonradioactive amino acids (in excess). The cells are then taken for protein extraction. Pulse-
chase analysis is used for the study of protein synthesis and processing, the examination of
intracellular localization of nascent proteins over time, and for the assessment of their secretion,
translocation, and degradation.

Apparatus

The pulse-chase experiment consists of three stages: the actual pulse-chase,

immunoprecipitation, and sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE). In the experiment, a fluorescently or radioactively tagged compound is used for time-
based monitoring of the cellular events. After the pulse-chase, the proteins of interest are
harvested by cell lysis through immunoprecipitation. The proteins are then run on SDS-PAGE
for further analysis. The equipment needed for the experiment includes an incubator, Eppendorf
tubes, a 26-gauge needle, a syringe, heat block, gel dryer, and Phosphor imaging screen.

 
 Radioactive pulse-chase

1. Incubate the MDCK cells (grown and infected with 10 infectious doses of influenza A virus per
cell for 5 hours at 37oC) with 5 ml Trypsin-EDTA at 37oC for 5 minutes.
2. Transfer the cells to a 50 ml BD falcon tube and wash them twice with 10 ml pre-
warmed Dulbecco’s phosphate-buffered saline (DPBS) and centrifuge for 1 minute at 2500 x g.
3. Resuspend the cells in 1 ml DPBS and transfer in a 1.5 ml microcentrifuge tube.
4. Centrifuge the cells at maximum speed for 15 seconds.
5. Resuspend the cells in 200 µl of pre-warmed pulse medium (Dulbecco Modified Eagle Medium
DMEM supplemented with 0.20 mM L-cysteine and 0.2 mCi/ml -methionine). Then, incubate
the cells in a water bath at 37oC for 2 minutes.
6. Centrifuge for 15 seconds at 4oC at maximum speed.
7. Resuspend the pellet in 1.05 ml pre-warmed chase medium (DMEM media supplemented with
7.5% fetal bovine serum and 67 mM L-methionine).
8. Incubate the cells in a water bath at 37oC for 20 minutes.
9. Take 190 µl aliquots each at 0, 5, 10, 15, 20 minutes.
10. Immediately transfer them to 1.5 ml centrifuge tubes containing 1 ml ice-cold DPBS.
11. Centrifuge for 15 seconds at 4 o
12. Resuspend the pellet in 1 ml non-denaturing lysis buffer (ice-cold)
13. Incubate the cell lysate at 4oC for 30 minutes with slow rotation.
14. Centrifuge for 15 minutes at 4oC at maximum speed.
15. Discard the pellet and keep the supernatant.

Immunoprecipitation
1. Wash 30 µl of protein A sepharose twice with 0.5 ml ice-cold Dulbecco’s phosphate-buffered
saline DPBS and centrifuge for 1 minute at 3000 x g at 4o
2. Resuspend resin in 0.5 ml ice-cold DPBS supplemented with 0.001% Triton X-100 and anti-HA
antibody of interest.
3. Incubate for 2 hours at 4oC under slow rotation.
4. Wash the resin with 0.5 ml ice-cold non-denaturing lysis buffer (0.5% Triton X-100, 50
mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA, and Complete mini, EDTA-free protease
inhibitor cocktail) for two times.
5. Add 10 µl bovine serum albumin.
6. Add cleared cell lysate from each aliquot.
7. Incubate for 2 hours at 4oC under slow rotation.
8. Wash the resin twice with 0.5 ml ice-cold non-denaturing lysis buffer supplemented with 0.1%
instead of 0.5% Triton X-100.
9. Wash the resin with 0.5 ml ice-cold DPBS.
10. Resuspend the resin in 20 µl of 4x LDS buffer.
11. Boil the samples for 5 minutes.

SDS-PAGE and fluorography

1. Load 15 µl of each sample on protein mini-gels.

2. Run the gel for 3 hours at a constant 50 mA/gel.
3. Fix the gels with 10 ml fixation solution (50% methanol and 10% acetic acid) for 30 minutes at
room temperature under slow rocking.
4. Dry gels in gel drier for 1.5 hours at 80o
5. Expose the films to the radioactive gels at room temperature overnight.

Single-cell pulse-chase for nuclear components 

Cell culture

1. Culture the cells in cell culture medium containing Dulbecco’s modified Eagle’s medium, 10
% fetal calf serum, nonessential amino acids, and penicillin and streptomycin (100 U/mL).
2. Grow the cells in an incubator at 37 °C with 5 % CO 2 supply and split them on 80 % confluence.
3. Plate the cells on specific media and perform transfections according to the standard protocols.
Applications

Analysis of the in vivo assembly of bacteriophage T4 tail 

The pulse-chase analysis is a non-invasive tool to determine the assembly pathway of the
complex tail of bacteriophage T4 virus. In the study, bacteriophage T4 mutants defective in the
head assembly were used to infect the E. coli cultures to study tail assembly in isolation. The
bacterial cultures were labeled with leucine on the onset of late protein synthesis. After the
complete tail began to accumulate at a constant rate, the bacterial cultures were pulsed with
methionine, and then chased. Completed tails were obtained and purified at one-minute intervals
for the next 30 minutes. It was found that the closer the assembly point to the end of the
pathway, the sooner the chase appears, presenting the assembly cascade. The results showed that
the tail completion proteins such as gp18 (tail sheath) and 19 (tail tube) show the earliest
inflection as compared to other tail proteins. The study validated the application of pulse-chase
analysis to analyze and assess the assembly of viral components.

Assessment of protein maturation and degradation 

The pulse-chase analysis is a powerful technique to analyze protein maturation and degradation.
Using the short pulses, the whole process of protein synthesis to degradation can be determined
in a natural environment. The technique has been widely used for endogenous and viral proteins
including thyroglobulin, vesicular stomatitis virus, influenza hemagglutinin, envelope proteins,
 and immunoglobulins. This biochemical approach is a cutting-edge analysis method to study a
variety of processes including protein folding, post-translational modifications, endogenous and
intracellular transport, and the rate of protein degradation.

Assessment of MHC class II biosynthesis, maturation, and peptide loading 

The pulse-chase analysis is a widely used technique for the exploration of the synthesis,
processing, and transport of proteins. In the research, the pulse-chase experiment was used to
study the major histocompatibility complex (MHC) class II synthesis, maturation, trafficking,
and peptide loading in human Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-
LCL). The MHC class II glycoproteins bind heterogeneous mixtures of peptides presented on the
surface of antigen-presenting cells for the inspection of CD4+ T helper lymphocytes. The results
obtained from these experiments can illustrate information to track changes in the molecular
associations, an abundance of radiolabeled proteins, and the conformation-sensitive monoclonal
antibodies (mAbs).

Biosynthesis, targeting, and processing of lysosomal proteins

The radioactive amino acids and modifier groups are incorporated into proteins to study their life
cycle and various modifications. Lysosomal enzymes can also be detected and characterized
using pulse-chase analysis. The pulse-chase labeling provides a detailed insight into organelle-
specific modifications of lysosomal proteins. For this, an antibody against lysosomal protease is
used as a reference. In the study, cathepsin Z was synthesized as a larger proenzyme containing
two N-linked oligosaccharides which mature to a shorter single-chain enzyme with
oligosaccharides. The pulse-chase experiment demonstrates the conversion of the precursor into
the mature form. The technique could also be used to study the deglycosylation of metabolically
labeled proteins and the alterations in the apparent size of the attached glycopeptides.

Precautions
 Prepare fresh pulse-chase media before use.
 During lysis, it is essential to maintain the nucleus intact.
 If the protein expression is relatively low, then the cells can be transfected with a plasmid
encoding the protein behind the promotor or a lipid mixture.
 Separate the folding process from translation if the effects of certain conditions such as ATP
depletion are tested.
 Cycloheximide should be added in chase medium if the kinetics of the process has to be studied.